Abstract:Proline with strong hydrophilic property is the only sub-amino acid being used for protein synthesis. The function and mechanism for proline has been extensively studied in plant. Proline is not only an osmolyte, but it also has important roles in scavenging reactive oxygen species, acting as a signal molecule to regulate growth, development, proliferation and cell death in plant. Recent studies have demonstrated that a proper amount of proline plays important roles in bacterial cells. The biosynthesis, degradation, intracellular and extracellular transport as well as the function of proline produced by bacteria were reviewed in this paper.

Abstract:As a ubiquitous phenomenon in eukaryotes, autophagy not only achieves the degradation and recycling of intracellular substances, but also interacts with a series of processes such as the appressorium development, turgor increase, mycelium formation, and completion of infection, etc, in the early infection stage of plant pathogenic fungi, and plays an important role in these bio-processes. In the present review the autophagy related genes and the autophagy process of phytopathogenic fungi are smmarized; the regulation and influence of autophagy on the growth and development and the pathogenicity of pathogenic fungi are generalized; the autophagy related signaling pathways of pathogenic fungi are illustrated; and the main molecular mechanisms of autophagy influencing the infection processes of phytopathogenic fungi are clarified. These provide new strategies and ideas for screening new drugs that inhibit pathogenic fungi infection by using autophagy-related genes or proteins as targets in future studies.

Abstract:Bacterial secretion systems can deliver specific effector molecular to outer environment or directly into target cells, resulting in the adaptive advantage from interaction between bacteria and host or bacteria in microbial communities. The type Ⅵ secretion system (T6SS) is a macromolecular secretory apparatus which is functionally and structurally analogous to the needle-like contractile tail of bacteriophages, wide-spreading in Gram-negative bacteria. It can translocate diverse enzyme and toxins of bacteria into eukaryotic or prokaryotic target cells in a contact-dependent manner. After delivery, the effector proteins can make an impact on both interbacterial competition and pathogenicity. In addition, some effectors can also enter the extracellular milieu in a non-contact dependent manner to help bacteria obtain scarce metal ions, which is essential for the maintenance of intracellular metal homeostasis under stress conditions. In this review, we summarize the advances in understanding the relevant aspects from the structure and assembly to the function of T6SS. We also focus on the broad area of T6SS effector and its mechanism of metal ion translocation, which contributes to our comprehension of the crucial role of T6SS in the microbial competition and host infection.

Abstract:As an important semiconductor, cadmium sulfide nanoparticles (CdS NPs) have outstanding photoelectric properties, adjustable band gap and chemical stability, and have a great application potential in related fields, such as:analytical chemistry, biomedicine, fluorescence imaging and biosensor. The biosynthesis of CdS NPs has been widely studied due to its controllable, low-cost and environmental friendly advantages. However, as a kind of nano-scaled metal sulfide materials, CdS NPs have severe toxic effects on prokaryotic microorganisms. This review summarized the research progress of the toxic mechanism of CdS NPs in prokaryotic cells, including the biosynthetic process of CdS NPs; the toxic effects of CdS NPs on E. coli cells and the defense mechanisms of the cells. Moreover, we discussed the toxic effects of different forms of cadmium on the biosynthetic cells during the CdS NPs biosynthetic process and the unique mechanisms of the NPs biosynthesis strains to respond to the cadmium stress. The purpose of this article is to evaluate the toxicity of CdS NPs on the bacteria cells in a more comprehensive way and to promote the application of the CdS-resistant prokaryotes in related fields.

Abstract:Although the relationship between hepatocellular carcinoma and intestinal microbiota has been experimentally and clinically confirmed, its ramifications regarding clinical diagnosis and therapy remain unknown. In this review, we elucidated the relationship between the intestinal microbiota and hepatocellular carcinoma as well as the obstacles to its use in the diagnosis of the latter. Moreover, we discuss the feasibility and usefulness of a novel comprehensive indicator of intestinal microbiota for the diagnosis of hepatocellular carcinoma, even though the bacterial taxa used in this indicator warrant further study. Finally, we provide suggestions for the screening of the bacterial taxa used in the calculation of this indicator.

Abstract:Arbuscular mycorrhizal fungi (AMF) are widely distributed in nature and can form mycorrhizal symbiosis with the roots of most vascular plants. They play important ecological roles in the regulation of plant community, and are deeply involved in the global carbon, nitrogen, and phosphorus cycling. They are also the most promising microbial groups in the fields of agriculture, forestry and environment. However, so far the information of their genomic and transcriptomic characters was limited, partially due to the technique limitations in their cultivation. In the past decade, researches on AMF genome and transcriptome have achieved a rapid development under the impetus of high-throughput sequencing. These studies have greatly improved our understanding of AMF in heredity, development, metabolic physiology and symbiosis mechanisms. Here we reviewed the research progresses in the available genomic and transcriptomic information of AMF based on published literature. We found that genomes of available AMF species commonly have large sizes, high transposon abundances, high GC contents, rich in functionally-unknown and species-specific genes, and are lack of some symbiosis-related genes. We also summarized the transcriptomic characteristics of AMF in different symbiotic structures, at different symbiosis stages and with different host plants. The results showed that the transcriptomic sizes generally varied among different AMF species. Also, in different symbiotic structures or at different symbiotic stages, diverse transcriptomic characteristics were found, especially for the expression profiles of genes related to nutrient exchange and signal transduction. In contrast, the transcripts of the same AMF in symbiosis with different host plants were relatively conserved. Finally, we proposed the research directions that need to be focused on in this field, including the innovation of AMF asymbiotic culture technology, the analysis of AMF gene functions, the study of non-model AMF groups and the study of AMF proteome.

Abstract:[Objective] The aim is to study the genotype diversity of indigenous Saccharomyces cerevisiae isolated from spontaneous fermentations of Vitis davidii Föex in Ziyun County of Guizhou Province, to analyze the dynamic changes of different S. cerevisiae genotypes at different fermentation periods, and to provide theoretical references for the industrial application of excellent S. cerevisiae resources. [Methods] We applied interdelta fingerprinting analysis and microsatellite marker analysis to study the genotype diversity of indigenous S. cerevisiae during spontaneous fermentations of Vitis davidii Föex in Ziyun County of Guizhou Province. DPS software was used to analyze the genetic relationship among different genotypes. [Results] We totally isolated 75 indigenous S. cerevisiae strains, and identified them into 10 genotypes by interdelta fingerprinting analysis and microsatellite marker analysis. Genotypes 6, 9, 10, 11, 14, 15 and 16 are the peculiar seven genotypes owned by indigenous S. cerevisiae strains. Genotypes 7, 17 and 18 are the mutual three genotypes owned by both indigenous and commercial S. cerevisiae strains. Furthermore, other commercial S. cerevisiae strains in the study showed nine other specific genotypes including genotypes 1, 2, 3, 4, 5, 8, 12, 13 and 19. The genotype 17 took the highest proportion in the 75 isolates (36%) followed by the genotype 10 (13.3%). Different genotypes showed waxing and waning ratio changes during fermentations, and the cell density of each genotype varied among 10⁴-10⁷CFU/mL. [Conclusion] Spontaneous fermentation samples of Vitis davidii Föex in Ziyun County of Guizhou Province showed rich genotype diversity of S. cerevisiae strains. Genotypes 10 and 17 were the predominant S. cerevisiae genotypes. This study laid a basis for the development of excellent indigenous S. cerevisiae resources of Vitis davidii Föexin Guizhou Province.

Abstract:[Objective] Based on the traditional relative quantitative method, the microbial community in pit clay with different cellar ages was investigated by using the absolute quantitative method with addition of an internal standard at the DNA extraction step. [Methods] According to the relative and absolute quantitative results, the microbial community and physicochemical factors of pit clay were profiled by using Clostridium acetobutylicum as an internal standard through 16S rRNA gene high-throughput sequencing. [Results] With the increase of cellular age, the diversity of microbial community in pit clay increased, and the relative abundances of representative genera such as Caproiciproducens, Methanobacterium, Methanosarcina, and Syntrophomonas significantly increased. By using the absolute quantitative approach, the biomass in the aged pit clay was 600 times and 28 times as much to that in the new pit clay and the aging pit clay, respectively. Compared with the relative quantification, on the contrary, the absolute abundance of Lactobacillus in aged pit clay did not decrease rather than increase during the aging process, with 20 times as that in new pit clay. The absolute abundance of Caproiciproducens in aged pit clay was 25000 times as much to that in new pit clay. Moreover, the absolute abundances of other functional microorganisms such as Methanobacterium, Methanosarcina, Methanobrevibacter and Syntrophomonas also increased across the aging process. The absolute abundance analysis indicates that the aging process of pit clay is a process with increased diversity as well as biomass of microbial community, contributing to lactate depletion and thus maintaining the stability of pit clay microbiota. [Conclusion] With the combination of relative and absolute quantitative approaches, the dynamic changes of species members in microbial community were profiled during the pit clay aging process. This study will help to provide a new quantitative and causal correlation analysis method to decipher the composition and function of pit clay microbiome.

Abstract:[Objective] The aim of this study was to screen the reference genes of Agaricus brasiliensis under different experimental conditions, especially under cadmium (Cd) stress, and to lay the foundation for studying the functional studies of cadmium enrichment-related genes in A. brasiliensis.[Methods] We screened 18 candidate genes and designed 30 primer pairs based on the transcriptomes of mycelia under the stress of different concentrations of Cd (0, 2, 5 mg/L). According to the specificity and amplification efficiency of primers, 5 pairs of primers from different genes were selected. The Ct values of mycelia, primordium, stipe, and pileus of 5 genes under different concentrations of cadmium stress were detected by QRT-PCR, and the expression stability of these genes was evaluated by geNorm, NormFinder, and BestKeeper. [Results] The results showed that Small glutamine-rich tetratricopeptide repeat-containing protein 2 (SGT2) and Serine/threonine-protein kinase (STK) were the two most stable candidate reference genes under Cd stress in the mycelial stage, GAPDH was the most stable reference gene in different tissues and SGT2, STK and GAPDH were the three most stable reference genes under all experimental conditions. [Conclusion] Under Cd stress conditions in A. brasiliensis, SGT2 and STK screened in this study were more stable compared to the commonly used reference genes.

Abstract:Dicholodiphenyltrichloroethanes (DDTs) is probably the best known and typical persistent organic pollutant in the world, which has been widely used in malaria control and agricultural deworming. They are still detected in various environmental matrices and has new input sources although their usage in agriculture has been banned in China and others. Numerous concerns have arisen over the past decades about the adverse environmental impacts(including harm to offshore ecosystem and human health) of DDTs. There has been a considerable interest over the last decades for the Rieske-type arylhydroxylating dioxygenases (RHDs) as they are seen as potentially capable of initiating their degradation. [Objective] In order to explore the degradation characteristics and mechanism of biphenyl dioxygenase(BPDO) on DDTs, we selected Burkholderia xenovorans LB400 biphenyl dioxygenase and its mutants to explore the degradation process of p,p'-DDT and o,p'-DDT. [Methods] Using BphAE_LB400 as parent, the mutant BphAE_S283M was obtained by two-step site-directed mutagenesis from Ser to Met. The degradation characteristics and mechanism of wild type and mutant were explored by comparing the catalytic performance of wild type and mutant to DDTs, simulating the structure of mutant protein and molecular docking. [Results] The data showed that BphAE_LB400 and BphAE_S283Mcould not be degraded p,p'-DDT, but BphAE_S283M metabolized o,p'-DDT and produced two stereoisomers. The structural analysis of BphAE_LB400 and BphAE_S283M showed that the reaction ring of p,p'-DDT did not coincide with the biphenyl reaction ring in the original crystal structure. In the o,p'-DDT-BphAE_S283M conformation, the proximal ring did not fit the biphenyl reactive ring as well, but its orientation toward the catalytic Fe²⁺ places two vicinal atoms at a distance(within 0.5 nm) that would allow a catalytic reaction. In addition, the surface area and volume of the catalytic cavity of BphAE_S283M is larger than that of BphAE_LB400, which is likely to contribute to the combination of BphAE_S283M and o,p'-DDT. [Conclusion] 283 is the key amino acid residue that affects the catalytic metabolism of BPDO to DDTs. It can affect the substrate specificity by adjusting the distance between the reaction carbon atom and the catalytic center and the size of the catalytic cavity. This will provide better insights about the bases for BPDO broad substrate range and about the mechanisms by which the enzyme evolves to change or expand its substrate range and its stereo- and regiospecificity.

Abstract:In China, the saline-alkali soil covers an area of approximately 99.13 million hm², of which the severe saline-alkali soil with pH value above 9 and salinity greater than 0.6% represents a growth rate of 1.4% per year. Improving plant rhizosphere environment by utilization of nitrogen-fixing microorganisms to increase crop yield is an important method for saline-alkali soil amelioration.[Objective] Candidate strains were selected for the amelioration of extreme saline-alkali soils by isolating the autogenous azotobacter from the soil sample collected from the coral reefs in Sansha City, Hainan Province. [Methods] The strains were identified through morphological observation, physiological and biochemical characteristics analysis and 16S rRNA sequencing, followed by the analysis of their nitrogen-fixing, salt-tolerant and growth-promoting characteristics, and verification of their effects on the main agronomic traits of corn. [Results] One strain of extremely salt-tolerant nitrogen-fixing bacteria named DJ-1 was obtained. The colony of the strain is round in shape,The individual bacterium is rod-shaped,sizing (0.3-0.5) μm×(0.5-1.3) μm. Negative in Gram Staining, the strain is highly homologous with the 16S rRNA sequence of Agrobacterium tumefaciens, hence it is determined to be Agrobacterium tumefaciens. Under medium culture condition, DJ-1 presents normal growth at pH value of 9 with NaCl concentration of 1%-4%, and tolerance at pH value of 12 and NaCl concentration of 8%. The nitrogenase gene nifH was cloned from DJ-1. It has been indicated in the pot experiment results that the growth of corn was significantly accelerated by DJ-1. [Conclusion] The strain DJ-1 has the tolerance to extreme saline-alkali conditions, coupled with high nitrogen fixation and growth-promoting capabilities. It may be a potential candidate strain of functional bacteria for poor saline-alkali farmland amelioration.

Abstract:[Objective] An intracellular persistence model of light chain of botulinum toxin type A was established to simulate long-term poisoning caused by botulinum toxin type A. [Methods] The recombinant plasmid pcDNA3.1-ALC-GFP was designed and constructed, the plasmid was extracted for PCR verification, and transfected into Nureo-2a cells for expression. Western Blot and cell immunofluorescence analysis were used to verify the expression of ALC-GFP and its long-term persistence in cells. An intracellular persistence model of light chain of botulinum toxin type A was established, and the model was used for the screening of anti-botulinum toxin drugs. [Results] The recombinant plasmid pCDNA3.1-ALC-GFP was successfully constructed and transfected into Nureo-2a cells, which verified the long-term persistence of ALC-GFP in cells. The intracellular persistence model of light chain of botulinum toxin type A was successfully established, and the potential anti-botulinum drug CB3 of botulinum toxin type A was screened by this model. [Conclusions] The intracellular persistence model of light chain of botulinum toxin type A was successfully established and applied to the screening of CS1, CE2 and CB3 drugs, which laid a foundation for the study of detoxification of botulinum toxin A long-term poisoning.

Abstract:[Objective] Morolic acid is derived from germanicol, has excellent anti-HIV and anti-inflammatory activities properties as oleanane-type triterpenoid, thus their potential applications in the pharmaceutical industry. In this study, synthetic biology was used to construct Saccharomyces cerevisiae cell factories for efficient biosynthesis of morolic acid. [Methods] Firstly, using CRISPR/Cas9 technology, three oxidosqualene cyclases (OSCs), namely MdOSC, PbOSC and RcOSC were integrated into S. cerevisiae to screen for high yield germanicol chassis cells. Then, to construct the engineered strain capable of morolic acid production, the cytochrome P450 oxidase (CYP716AL1) derived from Catharanthus roseus and the cytochrome P450 reductase derived from Jatropha curcas (JcCPR) were further integrated into S. cerevisiae. Moreover, to improve morolic acid biosynthesis, the CYP716AL1 was coexpressed with CPRs (AtCPR, LjCPR, GuCPR and MtCPR) derived from different plants. Finally, the mevalonate (MVA) pathway was modified by overexpressing the key pathway enzymes to improve morolic acid production. [Results] The engineered strain S201 obtained by integrating oxidosqualene cyclase MdOSC from Malus domestica yielded the highest amount of germanicol of 68.3 mg/L. The engineered strain S401 obtained by integrating CYP716AL1 and JcCPR in S201 achieved biosynthesis of morolic acid, and the yield reached 15.0 mg/L. Furthermore, the CYP716AL1 was coexpressed with AtCPR from Arabidopsis thaliana in S201, resulting in engineered strain S402, which yielded the highest amount of morolic acid of 24.3 mg/L. Finally, overexpression of key enzymes in the metabolic pathway of MVA in engineered strain S402, FPP synthase (ERG20) and squalene epoxidase (ERG1), resulted in a morolic acid yield of 34.1 mg/L (S6). [Conclusion] In this study, morolic acid cell factories were successfully constructed, which provides a theoretical and technical basis for the construction of high-yield oleanane-type triterpenoids cell factories.

Abstract:[Objective] The aim of this study is to explore the fungal community structures associated with rhizosphere soil, root and leaf of Stipa purpurea, and the relationship between rhizosphere soil fungal community structure and soil environmental factors. [Methods] We collected soil and plant samples from three different sites which located in the Qinghai-Tibetan Plateau. Soil physiochemical properties and enzymatic activities were detected by using the methods of soil chemistry, and rhizospheric and endophytic fungal community structures were analysed by Illumina Miseq high throughput sequencing technology. Based on this, we resolved the composition and richness, Alpha diversity and the characteristics of fungal community structures. Meanwhile, we also analyzed the correlation between the diversity of rhizosphere fungi and the soil environmental factors, and clarified the soil environmental factors which effected the rhizosphere mycobiota structure of Stipa purpurea. [Results] The results showed that rhizosphere soil pH were neutral or leaning alkalinity of three sampling sites, soil physiochemical properties and enzymatic activities changed differently in different sites. We obtained a total of 314801 effective sequences and 4611 OTUs by high throughput sequencing, the diversity and richness of fungal communities were the highest in GanSu site, and the lowest in XiZang site. The fungal communities' dominant phyla of all samples were Ascomycota and Basidiomycota, accounting for 88.28% of the total. The endophytic fungal communities had significant variations in different sampling sites, while rhizosphere soil fungal communities had no obvious differences in three sites. Correlation analysis showed that the diversity of fungal community was significant positive correlated with soil pH, the contents of available K, Fe, Ca, Mg, and soil peroxidase, soil polyphenol oxidase, soil dehydrogenase, while significant negative correlated with altitude and soil acid phosphatase. Redundancy analysis indicated that different fungal communities corresponding different impact environmental factors. [Conclusion] A wide variety of fungal communities are associated with the roots, leaves and rhizosphere soil of S. purpurea. The fungal assemblages are influenced by multiple soil environmental factors, and different fungal communities have different influencing factors. This study was significant to the exploitation of the beneficial microbial resource, and also providing a theoretical basis for the conservation and reasonable development these natural grasslands.

Abstract:[Objective] The distribution and physiological function of ε-poly-L-lysine-degrading enzyme (Pld) in Streptomyces albulus were investigated in this study. [Methods] The sequence of Pld in the reported ε-poly-L-lysine (ε-PL) producing strains were mined and analyzed from their genome by bioinformatics methods, and then two types of Plds in S. albulus M-Z18 genome were knocked out, complemented and overexpressed by genetic methods, those recombinant strains were used to study the degradation of ε-PL, the minimum inhibitory concentration (MIC) of ε-PL and their ε-PL productions. [Results] pldⅠ and pldⅡ are widely distributed in S. albulus, and their protein sequences are highly conserved. The results showed that PldⅠ and PldⅡ in the S. albulus M-Z18 all could degrade ε-PL. However, the degradation activity of PldⅡ was dominant, while PldⅠ and PldⅡ had a synergistic effect on the degradation of ε-PL. The MIC values of recombinant strains with pldⅠ or/and pldⅡ overexpression toward ε-PL were significantly increased, especially the MIC value of pldⅠ and pldⅡ co-overexpressed strain was 2.19 folds higher than that of the original strain. Surprisingly, these recombinant strains showed no significant difference in the production of ε-PL at pH 3.0-5.5 compared with the original strain. [Conclusion] PldⅠ and PldⅡ are highly conserved in S. albulus, which physiological functions are protect S. albulus from the inhibition of its metabolite ε-PL by degradation at the neutral pH.

Abstract:[Objective] Dibenzofuran (DBF) is a highly toxic compounds and widely spread in environment, used as a model compound in studying the microbial degradation of polycyclic aromatic compounds. [Methods] A bacterial consortium DBFC was isolated from petroleum contaminated soil in Liao River China. The total DNA was extracted to analyze the biodiversity of DBFC, and the strain was isolated and purified by plate dilution coating method. The optimum growth conditions of mixed bacteria were studied through measuring the absorption value of OD₆₀₀. The effects of substrate concentration, substrate, adding nutrient and surface-active substance on the degradation efficiency were studied under the optimum growth conditions. The intermediate metabolites of DBF degradation were detected by ultra-high resolution mass spectrometry, and the metabolic pathway was speculated. [Results] Microbial diversity analysis showed 84.06% of the strains identified in DBFC were Paenibacillus sp., 8.17% were Achromobacter sp., 0.77% were Pseudmononas sp., other strains contain only 2.13%. Four strains, belong to Ochrobactrum sp., Achromobacter sp., Stenotrophomonas sp. and Microbacterium sp., were isolated. Through cultivated in mineral-NH₄Cl salt medium adding DBF as the carbon source, the four stains could not grow. The optimal growth temperature and pH value was 30℃ and 8.0, respectively. Under optimal conditions, DBFC could completely degrade up to 1000 g/L of DBF in 8 days. The maximum degradation rate of DBFC was 0.031 mmol/(L·h) under 1.0 g/L of DBF. Adding glucose, yeast and peptone, the degradation rate of DBF were increased by 1.38, 1.14, and 1.24 fold, respectively. Adding sodium dodecyl sulfonate and Triton-X-100, the degradation rate were inhibited; while adding Tween-80, the degradation rate was decreased. Metabolites 2,2',3-trihydroxybiphenyl, 2,4-hexadienoic acid, gentisic acid and salicylic acid were identified by ultra-high performance liquid chromatography (UPLC). The bacterial consortium DBFC could also use gentisic acid and salicylic acid as sole carbon and energy source, which were respondence with the previous results. [Conclusion] The bacterial consortium DBFC had high efficient in degrading of DBF under pH 8.0 condition, which could be further exploited in situ remediation of polycyclic aromatic compounds contaminated sites. Through UPLC-MS/MS analysis, the metabolites were identified, which provides methods and directions for the study of metabolic process of the polycyclic aromatic compounds.

Abstract:[Objective] H. pylori adhesin A (HpaA) is a promising antigen for Helicobacter pylori vaccine. Adjuvant effects of chimeric flagellin cFLN and chitosan/tripolyphosphate (CS/TPP) nanogel encapsulation on humoral and gastric mucosal immune response induced by nasal vaccination of HpaA was investigated. [Methods] Chimeric flagellin cFLN was constructed by combining D0 and D1 domains of Salmonella typhimurium flagellin FliC with D2 and D3 domains of Helicobacter pylori flagellin FlaA. Chimeric flagellin cFLN was then linked with adhesin (HpaA) to construct a complex antigen cFLN-HpaA. cFLN, HpaA and cFLN-HpaA was expressed in recombinant E. coli and further purified by Ni-NTA Prepacked chromatographic column. cFLN, HpaA and cFLN-HpaA were further encapsulated into chitosan/tripolyphosphate (CS/TPP) nanogels to prepare cFLN-loaded CS/TPP nanogels (cFLN NG), HpaA-loaded CS/TPP nanogels (HpaA NG), and cFLN-HpaA-loaded CS/TPP/nanogels (cFLN-HpaA NG) by an ionic gelation method. In vivo immunogenicity studies were performed with mice. [Results] cFLN, HpaA and cFLN-HpaA was successfully prepared and encapsulated within CS/TPP/nanogels. After immunizing mice through the nose, compared with HpaA, cFLN-HpaA caused a 2.09, 1.4 and 2.62-fold increase in serum IgG, IgG1 and IgA levels, respectively. Compared with cFLN, HpaA and cFLN-HpaA, cFLN NG, HpaA NG and cFLN-HpaA NG increased the serum levels of IgA, IgG, IgG1 and IgG2a by 1.28 to 1.71 times. [Conclusion] cFLN and CS/TPP NG can significantly enhance the humoral immunity induced by HpaA delivered in the nasal cavity. The contents of IFN-γ, IL-4, IL-17 and SIgA in gastric mucosa after nasal immunization were detected. Compared with HpaA, cFLN-HpaA induced a 1.38, 1.16 and 1.58-fold increase in the content of IFN-γ, IL-4 and SIgA, respectively. Compared with cFLN-HpaA, cFLN-HpaA NG increased the contents of IFN-γ, IL-4, IL-17 and SIgA in mouse gastric mucosa by 1.81, 1.71, 2.16 and 2.10 times, respectively. It shows that cFLN and CS/TPP NG can significantly enhance Th1 and Th17 immune responses. In vivo immunogenicity studies in mouse indicated that cFLN and nanogel encapsulation could effectively not only enhance hurmoral immune responses, but also gastric mucosal immune response, Th1 and Th17 type immune response in gastric mucosa induced by nasally delivered HpaA. Therefore, these results suggested that cFLN-HpaA NG might be a promising nasally delivered vaccine system for protection against Helicobacter pylori infection.

Abstract:[Objective] In order to reduce the fermentation by-product vanillic acid, we established a CRISPR-Cas9 gene editing system for Amycolatopsis sp. to knock out the vanillin dehydrogenase gene (VDH). [Methods] Using VDH as the target gene, we replaced the promoters of tipA and j23119 on the pKCcas9dO plasmid with the Km^r promoter of the pRLE6 plasmid and the strong promoter permE* commonly used in Streptomyces, respectively. At the same time, we constructed the plasmid pKCKmCas9VDH by replacing the sgRNA with the specific sgRNA sequence that could recognize the target gene vanillin dehydrogenase, and linked the plasmid with the upstream and downstream homologous arms of the target gene to construct the knockout plasmid pLYZYP01. Then we transformed pLYZYP01 into Amycolatopsis sp. CCTCC M 2011265 and selected the VDH knockout mutant strains. [Results] We successfully obtained a knockout strain Amycolatopsis sp. ΔVDH with higher yield of vanillin. [Conclusion] We established a knockout system for Amycolatopsis sp. CCTCC M 2011265, and successfully knocked out the VDH gene. Under the condition of adding 12 g/L substrate ferulic acid, the yield of vanillin reached 9.19 g/L, and the conversion rate increased from 88.6% to 97.7%.

Abstract:LuxR family transcription factors can inhibit or stimulate the expression of various functional genes to maintain the stability of cell function. [Objective] To study the role of LuxR family transcription factors in Aeromonas hydrophila. [Methods] In this study, we have knocked out the AHA_1581 gene by homologous recombination technology to understand their role in virulence and drug resistance. [Results] The deletion of AHA_1581 gene not only enhanced the extracellular protease enzyme activity, the tolerance of A. hydrophila to survive under low temperature, different antibiotic stress, but also reduced the biofilm formation capability. Further the quantitative proteomic and bioinformatics analysis showed that the deletion of AHA_1581 gene 59 proteins were down-regulated and 142 proteins were up-regulated, which affected several important biological processes and virulence pathways in A. hydrophila. [Conclusion] Overall, the outcome of this study on the role of transcription factor AHA_1581 in the biological processes and virulence of A. hydrophila may pay the way for controlling their infections.

Abstract:[Objective] To investigate the prevalence, molecular characteristics and antibiotic resistance of oxacillin-susceptible, mecA-positive Staphylococcus aureus (OS-MRSA) isolates from pigs, we collected samples from 9 large-scale pig farms in four central and western provinces of China (Gansu, Shaanxi, Henan and Guangxi). [Methods] The OS-MRSA isolates were identified by amplifying nuc and mecA gene and oxacillin susceptibility tests. Twenty-six toxin-encoding genes, 16 kinds of commonly used antibiotic resistance and molecular typing (spa, MLST and SCCmec) of the isolates were detected.[Results] The results showed that 67 (7.6%, 67/884) of the 884 samples were contaminated with S. aureus. A total of 67 isolates were isolated, including 50 isolates of methicillin-susceptible Staphylococcus aureus (MSSA), 8 isolates of oxacillin-resistant mecA-positive (OR-MRSA) and 9 isolates of OS-MRSA. Among the 26 toxin-encoding genes, 9 toxin-encoding genes were detected, and the hla gene had the highest detection rate, followed by hld, hlb, hlg, sei, sem, seg, sen and seo. Sixteen isolates that carrying the enterotoxin-encoding genes, OR-MRSA and OS-MRSA isolates accounted for 37.5% (6/16) and 50.0% (8/16), respectively. The isolates carrying the enterotoxin-encoding genes were all ST9-t899 clone type. Among the 16 antibiotics tested, the isolates were resistant to 12 antibiotics, among which MSSA, OR-MRSA and OS-MRSA isolates were mainly resistant to 1-8, 10-12 and 7-11 antibacterial drugs, respectively. Four clone types (ST398-t571, ST9-t899, ST398-t034 and t11241) were identified, among which ST9-t899 was the only clone type of MRSA and ST398-t571 was the dominant clone type of MSSA. With the exception of four isolates where SCCmec typing was not detected, IV_b (76.5%, 13/17) was the only type in MRSA isolates. [Conclusion] To sum up, the sensitivity of pig MRSA isolates to oxacillin had changed, and they were generally sensitive to oxacillin. In addition, the dominant clone types of MSSA and MRSA strains were ST398-t571 and ST9-t899, respectively. The study also found that the clone type is significantly related to the toxin genes carrying status, and the clone type of the strain carrying the toxin genes was ST9-t899. Therefore, understanding the prevalence, molecular characteristics and drug resistance of MSSA and OS-MRSA from pigs in China can provide data support for the prevention and control of Staphylococcus aureus infection for pigs.

Abstract:[Objective] To investigate the activity of the peptide probrelin from traditional Chinese medicine grub against Candida albicans. [Methods] The broth microdilution method was used to determine the MIC of probrelin against normal and clinical resistant strains of C. albicans, and the MFC were determined by plate count method. Time-kill curves were drawn by plate counting after treatment with different concentrations of probrelin. The influences of probrelin on the integrity of the cell membrane and morphology of C. albicans were evaluated by PI absorption experiment and scanning electron microscope, respectively. The binding effect between probrelin and nucleic acid was evaluated by DNA gel retardation assay. Crystal violet staining method was used to evaluate the influences of probrelin on the formation of biofilm and mature biofilm of C. albicans. And the influences of probrelin on the hyphae formation of C. albicans were also tested. Checkerboard method was used to determine the interaction between probrelin and antifungal drugs. And a mouse subcutaneous infection model was used to tested the activity of probrelin against C. albicans under physiological conditions. [Results] The MICs of probrelin against normal and clinical resistant strains of C. albicans were 100 μg/mL, and the MFCs were 100-200 μg/mL. Probrelin showed time-and concentration-dependent antifungal activity against C. albicans, as well as on the integrity of the cell membrane, and could destroy the cell wall, but did not bind with nucleic acids. Probrelin not only inhibited the formation of the biofilm of C. albicans, but also disrupted the mature biofilm, and could also inhibit the hyphae formation. Furthermore, probrelin showed synergistic effect with the antifungal drug Clotrimazole, and could effectively clear C. albicans cells in the mouse subcutaneous infection model. [Conclusion] Probrelin showed good activity against C. albicans, and had the potential for the development of anti-C. albicans drugs.

Abstract:Natural products from marine fungi have been attracting attention for their unique structures and biological activities. Genome mining has become an emerging strategy to explore their metabolites. [Objective] In this study, we investigated Parengyodontium album SCSIO SX7W11, a marine fungus derived from South China Sea corals for the potential of producing polyketides. [Methods] We sequenced the whole genome of strain SX7W11 using Illumina Miseq. The polyketide biosynthetic gene clusters were then predicted and the functions of open reading frames (ORFs) were annotated. Normal phase chromatography, reversed-phase chromatography, Sephadex LH-20 chromatography, and High Performance Liquid Chromatography (HPLC) were conducted for the isolation and purification of natural products. The purified compounds were characterized by high resolution mass spectrometry (HR-ESI-MS), ¹H NMR, ¹³C NMR and X-ray data and the biosynthetic pathways were deduced. [Results] The whole genome scanning and sequencing results showed that the genome length of SX7W11 is 34.0 Mb, containing 24 biosynthetic gene clusters. Three polyketides were identified as emodin (1), alternaphenol B (2), and sydowinin A (3). The crystal data of sydowinin A was obtained. The biosynthetic gene cluster of sydowinin A was localized from SX7W11 genome by bioinformatics analysis. The biosynthetic pathway was then proposed. [Conclusion] Through genome mining and medium optimization, P. album SCSIO SX7W11, a coral derived fungus, has shown the potential to produce sydowinin type polyketides. This work laid a foundation for the biosynthesis study of sydowinin.

Abstract:[Objective] To compare the differences in gut microbiota in 34-week-old infants under continuous breastfeeding with different delivery modes, and to explore the impact of delivery modes on the development of infant gut microbiota.[Methods] Healthy full-term breast-fed infants were recruited and 21 infants were still participating in follow-up at 34 weeks, including 16 infants delivered by cesarean and 5 vaginal delivered infants. The 16S rRNA of the gut microbiota was detected. [Results] The two groups of 21 samples contained 6 phyla: Actinobacteria, Bacteroides, Firmicutes, Fusobacteria, Proteobacteria and Verrucomicrobia; 57 genera were found. Among them, there are 26 genera in Firmicutes, 18 genera in Proteobacteria, 6 genera in Actinomycota, 5 genera in Bacteroides, 1 genera in Fusobacteria and 1 genera Verrucomicrobia. Among them, the content of Proteobacteria in the gut microbiota of the vaginal delivery group (44.17%) was higher than that in the cesarean section (16.10%); and the content of Actinomycetes in the gut microbiota of vaginal delivery infants (0.00%) was lower than cesarean delivery babies (0.09%). At the genus level, compared with the cesarean section group, the abundance of a total of 7 microorganisms in the vaginal delivery group was significantly reduced (P<0.05), namely Staphylococcus, Lactobacillus, Parabacteroides, Enterococcus, Haemophilus, Bifidobacterium and an OTU of Enterobacteriaceae. [Conclusion] The mode of delivery has an impact on gut microbiota of infants continued breastfeeding, and this impact still exists at 34 weeks after birth.

Abstract:[Objective] The emergence of durg-resistant Mycobacterium tuberculosis brought a tough challenge to tuberculosis treatment. [Methods] Considering the lack of homologous recombination in Mycobacterium tuberculosis's genome, the mutation strongly related to resistance could be efficiently confirmed by genome-wide association analysis. However, many resistance-related mutations had yet been found by existing methods. To calculate resistant-related mutations, researchers commonly used some methods similar to genome-wide association analysis (GWAS), which mainly included genome-wide efficient mixed model association (GEMMA), phylogenetic convergence test (phyc), plink. To find the better method among the three methods when calculating resistant-related SNPs on non-mobile antibiotic resistance gene, the genomes of 1504 M. tuberculosis strains from Hunan province and National Center of Biotechnology Information was obtained with their phenotypes of three first-line antibiotics (isoniazid, rifampicin, ethambutol). The three methods were performed to calculate the association between phenotype and known or novel SNPs related to resistance, and their sensibility and specificity were evaluated by the resistant-related SNPs got by the three methods. [Results] Phyc was able to search more known resistance-related SNPs with the sensibility and specificity higher than 52.49%.[Conclusion] Phyc is the most accurate in predicting the SNP related to drug resistance of Mycobacterium tuberculosis, but considering the update of running time and phenotypic data, the results of GEMMA and plink should also be used as a reference.

Abstract:[Objective] Identification of regulatory factors for acarbose biosynthesis and harnessing them for the improvement of acarbose yield in Actinoplanes sp. SE50/110. [Methods] Firstly, regulatory proteins binding to the two bi-directional promoters of acarbose biosynthetic gene cluster were obtained using DNA affinity chromatography. Secondly, to validate functions, these coding genes of regulatory proteins were deleted or overexpressed in Actinoplanes sp. QQ-2. Next, soluble proteins were obtained by heterologous expression in E. coli BL21(DE3), and electrophoretic mobility shift assays were performed to verify the interaction between these regulatory proteins and promoter regions. [Results] By analyzing the results of affinity chromatography and mass spectra, we identified nine regulatory proteins (ACPL_1889, ACPL_4236, ACPL_7303, ACPL_6479, ACPL_8104, ACPL_8270, ACPL_5445, ACPL_3989, ACPL_7617). Furthermore, we studied the potential function of all the nine regulatory proteins by deleting or overexpressing their coding genes in the strain QQ-2. The overexpression of ACPL_1889 resulted in 25% yield increase, whereas its deletion led to 22% yield decrease of acarbose. Respective overexpression of ACPL_5445 and ACPL_3989 resulted in 12% and 39% yield decrease, whereas their deletions let to 15% and 8% yield increase, respectively. Meanwhile, transcription level of acarbose biosynthetic genes acbA, acbB, acbW and acbV increased when ACPL_1889 was overexpressed and decreased when it was deleted; the transcription of these four genes increased to a certain extent in ACPL_5445 mutant; whereas the transcription of these four genes decreased in the ACPL_3989-overexpressed mutant, the transcription of acbW and acbA increased by 100 times and 40 times in the ACPL_3989 deleted mutant, respectively. Moreover, we found both ACPL_1889 and ACPL_3989 were able to bind to promoters of the acb gene cluster in EMSA experiments. Eventually, we increased the yield of acarbose by 32% applying a combinatory strategy of overexpressing positive regulatory genes and deleting negative regulatory genes. [Conclusion] This study identified nine regulatory proteins binding to the two bi-directional promoters of acarbose biosynthetic gene cluster, among which ACPL_1889 is a positive regulatory factor, while ACPL_5445 and ACPL_3989 are negative regulatory factors. This work not only laid a foundation for studying the regulatory mechanism of acarbose biosynthesis, but also substantially improved acarbose yield by manipulating these regulatory genes.

Abstract:[Objective] Stellera chamaejasme L., a perennial herb of Stellera, is one of symbolic plants of grassland degradation in China. The purpose of this study was to explore the microbial community structure in the rhizosphere of Stellera chamaejasme L. and its relationship with soil environmental factors and enzyme activity in Gansu alpine steppe, so as to provide a theoretical basis for controlling grassland degradation caused by Stellera chamaejasme L. invasion. [Methods] The Illumina MiSeq high-throughput sequencing technology was used to analyze the composition and diversity of rhizosphere soil microbes of Stellera chamaejasme L. in various regions of Gansu, and further analyze the relationship between soil physical and chemical properties, enzyme activities and microbial communities. [Results] The results indicated that the pH value of the rhizosphere soil of Stellera chamaejasme L. in different areas increased with altitude, showing an upward trend. The content of macro and micronutrient elements in the soil, and the changes of soil enzyme activities were different. At the phylum level, Ascomycota, Zygomycota and Basidiomycota are dominant in rhizosphere soil fungi. Actinobacteria, Proteobacteria and Acidobacteria belong to the dominant groups in rhizosphere soil bacteria. The numbers of fungi and bacteria OTU (Operational taxonomic unit) and Shannon diversity index of altitude 2964 m are higher than the other four sample points (altitude 2373 m, 2608 m, 2733 m, 3280 m). Redundancy analysis showed that the soil microbes in the rhizosphere of S. chamaejasme are different and are affected differently by soil environmental factors. The correlation coefficient showed that the diversity of soil fungi is positively correlated with soil potassium, phosphorus, iron, calcium, molybdenum, altitude, soil moisture, and peroxidase, as well as soil temperature, polyphenol oxidase, dehydrogenase, invertase, and alkaline phosphatase is negatively correlated. Soil bacteria are negatively correlated with soil pH, potassium, phosphorus, magnesium, calcium, and soil moisture, and positively correlated with polyphenol oxidase, peroxidase, urease, dehydrogenase, invertase, acid phosphatase, and alkaline phosphatase. [Conclusion] The composition and diversity of the rhizosphere microbial community of S. chamaejasme in the alpine grasslands of Gansu Province are obviously different. In addition, the physical and chemical properties of soil have a strong positive correlation with soil fungi, and the soil enzymes have more influence on the composition and diversity of soil bacterial communities.

Abstract:[Objective] By using comparative proteomics, we identified several potential T6SS-2 effectors, including VP2918. The aim of this study is to elucidate the role of VP2918 in biological characteristics and pathogenicity of Vibrio parahaemolyticus. [Methods] The vp2918 gene deletion mutant strain (Δvp2918) and complementary strain (CΔvp2918) were constructed via homologous recombination technology. Subsequently, the growth characteristics, biofilm formation ability, motility, adhesion and cytotoxicity to HeLa cells, and mice lethality rate, bacterial colonization were analyzed in the wild-type strain (WT), Δvp2918 and CΔvp2918. [Results] The biological characteristics analysis in vitro showed that there was no significant difference in growth curve, motility, biofilm formation ability and adhesion ability to HeLa cells among WT, Δvp2918 and CΔvp2918. However, compared with WT, the toxicity of Δvp2918 to HeLa cells was significantly weakened. Animal experimental results showed that compared with the mice infected with WT, the mice infected with Δvp2918 showed slighter symptoms and higher survival rate. The bacterial loads of Δvp2918 were significantly lower than that of WT in spleen and liver of infected mice, whereas the complementation strain restored the virulence to resemble that of WT. [Conclusion] The VP2918 was not associated with the motility and biofilm formation ability, but it was a potential virulence factor and associated with the pathogenic of V. parahaemolyticus.

Abstract:[Objective] This study aims to investigate the effects of Lactobacillus reuteri strain DSM (LR) and Lactobacillus taiwanensis strain BCRC (LT) on intestinal inflammation using a mouse model of dextran sodium sulfate (DSS) induced colitis. [Methods] Twenty-four 8-week-old C57BL/6J male mice with similar body weight were selected and randomly divided into 4 groups (6 replicates in each group):normal group, DSS group, LR bacteria treatment group and LT bacteria treatment group. After 7 days adaptation period and 16 days experiment period, the mice were sacrificed and sampled. [Results] Compared with the normal group, the body weight and colon length of the DSS group were significantly reduced, and the disease activity index (DAI) scores as significantly increased (P<0.05). There was no significant difference in body weight between the LR and LT bacteria treatment groups and the DSS group (P>0.05), whereas the LR and LT bacteria treatment significantly reduced the DAI scores and the LT bacteria treatment treatment increased the colon length. The colonic histopathological scores of the normal group and the LT bacteria treatment group were significantly lower than those of the DSS and LR bacteria treatment groups (P<0.05). Colonic myeloperoxidase (MPO) activity was also significantly lower in the normal group and the LT bacteria treatment group than the DSS group, and there was no significant difference between the LR bacteria treatment group and the DSS group (P>0.05). Compared with the normal group, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon and plasma of the DSS group were significantly increased, while the levels of the anti-inflammatory factor IL-10 were significantly decreased. Compared with the DSS group, LT bacteria treatment group significantly reduced the levels of TNF-α, IL-1β, and IL-6 in the colon and plasma, and increased the level of IL-10; the LR bacteria treatment group significantly reduced the levels of colonic TNF-α and plasma IL-1β, and increase the level of IL-10 (P<0.05). The results of immunohistochemistry and ELISA showed that compared with the normal group, the DSS group reduced the expression levels of colonic tight junction proteins including Claudin-1, Occludin, ZO-1, and mucin Muc-2, while the treatment of LR and LT bacteria up-regulated the expression of those barrier-related proteins under the influence of DSS. The composition of colonic flora was detected by using 16S rDNA sequencing technology. PCoA analysis showed that the normal group and the DSS group are significantly separated, and the flora composition of the LT treatment group was closer to the normal group. At the phylum level, the abundance of Proteobacteria in the normal group was significantly lower than that of the DSS group (P<0.05). Compared with the DSS group, the LT bacteria treatment group could also reduce the abundance of Proteobacteria, but did not reach a significant level (P>0.05). At the genus level, Muribaculaceae_norank in the normal group was significantly higher than the other three groups, and Bacteroides in the normal control group was significantly lower than the other three groups (P<0.05). Compared with the DSS group, the treatment of LR and LT bacteria increased Muribaculaceae_norank abundance, and reduced Bacteroides abundance, but the difference was not significant (P>0.05). [Conclusion] Lactobacillus reuteri and Lactobacillus taiwanensis can strengthen the intestinal epithelial barrier and relieve the intestinal inflammation caused by DSS to a certain extent. Among them, Lactobacillus taiwanensis has a more significant protective effect on the intestinal epithelial barrier.

Abstract:[Objective] To investigate the effects of Na₂SeO₃ and Epichloë sp. on Festuca sinensis, we studied the growth indexes and metabolic compounds. [Methods] By using gas chromatography-mass spectrometer, we detected the metabolic compounds of the shoots and roots of F. sinensis two geographical populations with and without Epichloë endophyte, and metabolite differences were determined among treatments using LSD test. [Results] Geographical populations Haibei (HB) and Yushu (YS) respectively had 206 metabolites and 205 metabolites. There were significant differences in metabolites between shoots and roots. Geographical population HB shoots, HB roots, YS shoots and YS roots, respectively showed 27, 42, 40 and 33 marked metabolites using LSD (P<0.001). These marked metabolites were some nitrogen compounds, carbohydrates and organic acids. 9, 8, 5 and 14 common marked metabolites were respectively found between HB shoots and roots, between YS shoots and roots, between HB and YS shoots, and between HB and YS roots. Epichloë endophyte caused a highly significant increase in tyrose for geographical population HB shoots and roots (P<0.001). Selenium induced highly significant increases in α-ketoisocaproic acid in HB E+ and E-shoots and roots (P<0.001). There were highly significant increases of aspartic acid for HB shoots and roots by interaction of the Se and Epichloë endophyte (P<0.001). In addition, selenium treatment highly significantly increased α-ketoisocaproic acid and acetol for shoots and roots in geographical population YS E+ and E-(P<0.001). [Conclusion] There were significant differences in metabolites of the shoots and roots of two geographical populations. Epichloë endophyte infection and/or selenium treatment can increase the concentrations of some compounds.

Abstract:[Objective] Antarctic environments are extremely diverse and affected by human activities in varying degrees. The aim of this study is to explore distribution and migration of soil antibiotic resistant genes (ARGs) in different latitude regions of Antarctica.[Methods] We selected soil metagenomic data from Antarctic high (HLG) and low latitude regions (LLG) and surrounding regions of feedlot. Metagenomic reads was assembled using software MetaWRAP. ARGs, plasmids and integrative and conjugative elements (ICEs) were annotated using CARD (The Comprehensive Antibiotic Resistance Database), PlasFlow and ICEberg, respectively. [Results] In the Antarctic soils, Proteobacteria, Actinomycetes, Bacteroidetes and Firmicutes were the dominant phyla. Belonging to 25 ARG types, 406 ARGs were found, among which multidrug, tetracycline and aminoglycoside resistance genes dominated. The NMDS analysis showed that the characteristics of ARGs in Antarctic soils were significantly different from those in agricultural soils nearby feedlot (ANOSIM, P=0.001). The proportion of ARGs in all ORFs in the Antarctic HLG was 0.28%, lower than that in the LLG (1.93%, P<0.01). Different ARGs present different latitude-region resistance patterns. Therein, nitroimidazole, aminoglycoside, glycopeptide and macrolide resistance genes were mainly distributed in the HLG, while tetracycline and sulfonamide resistance genes were mainly distributed in the LLG (P<0.05). The migration analysis of ARGs in Antarctic soils showed that 17% of the detected ARGs were carried by plasmids. In addition, 163 ICEs carrying ARGs, belonging to 14 ARG types (e.g., multidrug, peptide and tetracycline resistance genes), were found. These ICEs carrying ARGs were extensively distributed among α-, β- and γ-proteobacteria. [Conclusion] The differences in microbial communities and ARGs between the Antarctic high and low latitude areas occurred, plasmids and ICEs facilitated migration of nature ARGs. This study provided a certain data basis for understanding the natural antibiotic resistance from the pre-antibiotic era.