Abstract:Bacteria have to adapt complex environment for survival in nature. For this environmental fitness, bacteria generate different response mechanisms to perceive environmental signals for regulating cell physiological and biochemical reaction in the long-term evolution process. Gene expression regulation is one of the response mechanisms with the transcription level and the post-transcriptional level. Non-coding RNAs (ncRNAs) play an important role in the post-transcriptional regulation in bacteria. Hfq is required for the action of many ncRNAs that act by base-pairing with target mRNAs. This review summarizes the function of ncRNAs and Hfq in post-transcriptional regulation, including:1. the classification and characteristics of ncRNAs; 2. the structure and function of Hfq protein; 3. non-coding RNAs and Hfq interaction.

Abstract:Gram negative bacteria contain two layers of membranes:Inner membrane and Outer membrane. Lipopolysaccharides of most Gram-negative bacteria are the most important component of membrane lipid and anchored in the outer leaflet of the outer membrane, which play a role in inherent immune of Gram-negative bacteria. Lipopolysaccharide transport system assembles lipopolysaccharides into the outer membrane to achieve a variety of functions, including lipopolysaccharide associated barrier, organic solvent tolerance, hydrophobic antibiotic resistance, membrane permeability and other functions. The transport system mainly depends on 7 different lipopolysaccharides transport proteins (LptABCDEFG), which are composed of the ATP-binding cassette transporter complex LptB2FG, periplasm protein LptA/C, and "vital gate" outer membrane protein complex LptDE together. This paper describes the structure and functions of lipopolysaccharide transport system in Gram-negative bacteria, to provide information to further study the function of each protein in this system.

Abstract:Many polyketides isolated from marine sponges are generated by symbiotic microorganisms via trans-AT polyketide synthases. Cloning the biosynthetic gene clusters of biologically active polyketides using metagenomics not only elucidates the biosynthetic pathways of bioactive compounds, but also makes it possible to obtain the target compounds by heterologous expression. This review presents recent progresses on the biosynthesis of polyketides derived by trans-AT polyketide synthases (PKSs) and related biosynthetic pathways in marine sponges.

Abstract:Plant terpenoids are the natural secondary metabolites derived from units of isoprene with a molecular formula of C₅H₈ from different types of plants. D-Limonene is recognized as monoterpene and widely used in food and medical industry because of many functions, such as anti-bacteriostasis, aroma enhancement, anti-cancer and anti-cough. At present, the industrial production of D-limonene is generally obtained by extraction from the peel or pulp of plants. However, the extraction of D-limonene from plants suffers from complex separation and purification, low efficiency and high energy consumption. At the beginning of this century, the rise of synthetic biology technology has brought new ideas and tools for the synthesis of natural active compounds, which has broken the boundary between species and has become a reality of synthesis of D-limonene from microbes. It is of great economic and social benefit to construct a targeted and efficient microbial cell factory for the synthesis of D-limonene, and to replace the traditional method of plant extraction with microbial fermentation. We reviewed recent achievements of metabolic engineering of Saccharomyces cerevisiae to synthesize terpenoids and elaborated Saccharomyces cerevisiae as microbial chassis, using the method of metabolic engineering and synthetic biology to build high heterologous production of D-limonene synthetic strategies.

Abstract:[Objective] A marine Vibrio X511 with strong ability to use alginate was isolated from the rotten Sargassum in the Weizhou Island of the North Sea. The metabolic pathway of using alginate by Vibrio X511 was studied by transcriptome sequencing.[Methods] Transcriptions sequencing was done by Illumina HiSeq2500 sequencing platform and the transcriptome of the strains cultured in alginate and glucose were sequenced. Differential transcripts were compared and analyzed, and the sequencing results were confirmed by fluorescence quantitative PCR. The fluorescence quantitative PCR was applied to verify the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to annotate the function and pathway in differential transcripts.[Results] There were 2024 differentially expressed genes in the culture of alginate compared to those in glucose, with 1066 genes up-regulated and 958 genes down-regulated. Some genes in the ubiquitous metabolic pathway were also differentially expressed in different cultures. The transcriptions of all the genes involved in alginate utilization and the key genes of synthetic ethanol in Vibrio X511 were up-regulated. In addition, the strain had a unique way to utilize alginate and one of the metabolic processes has not been reported in Vibrio.[Conclusion] Our findings enrich the research of biological method for degradation of alginate, and provide valuable data for the research of large seaweed biomass-based energy.

Abstract:[Objective] Microbial activities are considered the main cause of most food spoilage. Study of the regulation mechanism of spoilage is valuable to maintain quality and safety of food. Pseudomonas fluorescens is a typical Gram-negative spoilage bacterium. The aim of this study was to elucidate the role of the alternative sigma factor RpoS in spoilage caused by P. fluorescens.[Methods] An rpoS mutant of P. fluorescens isolated from refrigerated fish was constructed by homologous recombination. Survival rates of the wild type and the mutant under different stress conditions were compared. The types and contents of extracellular autoinducers acylated homoserine lactones (AHLs) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the wild type and the mutant were inoculated into the sterile salmon juice stored at 4℃, and total viable count (TVC) and total volatile basic nitrogen (TVB-N) were assayed.[Results] The in-frame deletion rpoS mutant strain was successfully constructed in P. fluorescens. Deletion of rpoS gene significantly decreased the resistance to 10 mmol/L H₂O₂and 15% alcohol, increased the resistance to 150 μg/mL crystal violet and 175 mmol/L acetic acid, and had no influence on the resistance to 47℃ and 20% NaCl. The ropS mutant produced higher contents of long chain AHLs C₁₀-HSL, C₁₂-HSL and C₁₄-HSL than the wild type one. In addition, the mutant reduced TVB-N production in sterilized salmon juice stored at 4℃.[Conclusion] These results indicated that RpoS was not only involved in the resistance to multiple stresses, but also influenced quorum sensing and spoilage potential of P. fluorescens.

Abstract:[Objective] To isolate and identify Bacillus spp. to control Neocosmospora pod rot of peanut (NPRP).[Methods] Bacillus spp. of peanut rhizosphere soil were isolated to inhibit Neocosmospora vasinfecta by plate confrontation method, and further classified by multiphase classification method. The lipopeptide synthesis gene type of biocontrol bacteria was detected by PCR. The control effect of Bacillus spp. on NPRP was tested in field trial.[Results] Twenty-eight Bacillus spp. strains were isolated and eight obviously inhibited N. vasinfecta. Two were identified as B. subtilis and the others were B. amyloliquefaciens. The biocontrol bacteria harbored one type of lipopeptide synthesis genes at least and all harbored FenB gene. We speculated that the inhibitory mechanism of the Bacillus spp. may be related to the synthesis of lipopeptide antibiotics. B. amyloliquefaciens GF-3 and GF-22 both could effectively reduce the disease index of NPRP and increase the yield of peanut in field trial.[Conclusion] Two strains B. amyloliquefaciens were isolated and identified to control NPRP. This study could provide strains to make biocontrol agents for prevention and control NPRP.

Abstract:[Objective] The aim of this study was to purify and characterize a dual-domain β-propeller alkaline phytase (phyHT) from Bacillus sp. HJB17 and to analyze the sequence features for application of the enzyme.[Methods] The amino acid sequence of phyHT was analyzed by bioinformatics methods. PhyHT protein was expressed, refolded, purified and the enzymatic properties were determined by ferrous sulfate and molybdenum blue method.[Results] Sequence analysis suggested that phyHT consisted of 633 amino acids and contained 2 phytase domain and belonged to typical BPP phytase. PhyHT was a hydrophilic protein and the molecular weight and theoretical isoelectric point were 69321.68 Da and 5.37, respectively. In the secondary structure, phyHT mainly consists of α-helix and β-sheet. Its three-dimensional structure mainly consists of β-sheet propeller. The optimum temperature of phyHT was 37℃, and stable at 45℃. PhyHT has an optimum pH of 8.0, and stable between pH 6.0 and 12.0. Low concentration of Ca²⁺ and Mg²⁺ promoted the enzymatic activity, whereas phytase activity was strongly inhibited by Fe²⁺, Mn²⁺, Zn²⁺, Cu²⁺ and Ni²⁺.[Conclusion] phyHT has important applications in theoretical research and in industry.

Abstract:[Objective] To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application, we induced Pseudomonas aeruginosa resistance to tachyplesin I and studied the preliminary resistance mechanisms, which may provide theoretical basis for the widely application of tachyplesin-I.[Methods] First, we induced P. aeruginosa resistance to tachyplesin I using continuously increasing concentration selection pressure and monitored bacterial resistance. Second, we studied the stability, cross-resistance and cost of resistance of resistant strain. Last, we investigated the potential role of extracellular proteases, extracellular polysaccharide content and biofilm formation in the resistance mechanism.[Results] After more than 30 serial transfers in P. aeruginosa ATCC27853 under increasing concentrations of tachyplesin I selection, the MICvalues for P. aeruginosa was gradually increased, whereas high resistance to tachyplesin I was produced until 80 serial transfers. P. aeruginosa ATCC27853 showed resistance to tachyplesin I under long-term, continuously increasing concentration selection pressure. Cross-resistances between tachyplesin I, amikacin and other antimicrobial peptides (pexiganan, tachyplesin Ⅲ, and polyphemusin I) were observed in resistant mutant. Our resistant strain displayed a substantial cost of resistance mainly in the form of a much longer lag phase in the absence of tachyplesin I in P. aeruginosa, whereas in the presence of tachyplesin I, resistant strain had a shorter lag phase and greater growth rate. The resistant strain P. aeruginosa ATCC27853-88-2 exhibited increased levels of extracellular proteolytic activity and reduced the antimicrobial activity of tachyplesin I. Under the same conditions, extracellular polysaccharide content of the resistant strain was higher, more easily to form biofilm than the original strain.[Conclusion] We demonstrate that long-term continuous exposure to high concentrations of tachyplesin I can induce resistance in P. aeruginosa ATCC27853 and the potential involvement of extracellular protease and biofilm formation in mediating this resistance.

Abstract:[Objective] The cofactor-required prokaryotic expressions of extrinsic protein often show low activity. To improve the enzyme activity and reduce the cost of adding cofactors, we tried to express CAT-POD in Escherichia coli and promote the related synthetic metabolism of enzymes cofactors.[Methods] We cloned katG, the CAT-POD coding gene of Halomonas elongata DSM2581, and constructed it into the prokaryotic expression vector pet28a-katG, to achieve the recombinant expression of CAT-POD in E. coli. Additionally, CAT-POD's activity depends on its active centre, heme. And hematoporphyrin is the main structure of the heme. We cloned hemA, a gene coding 5-amino levulinic acid synthetase, and constructed it into the prokaryotic expression vector pUC19-tac-hemA. By overexpressing hemA, we raised the content of the porphyrin in E. coli, and improved the enzyme activity of recombinant protein CAT-POD.[Results] Ultimately, the CAT enzyme activity of katG reached 377 U/mL, 7.5 times of the control group.[Conclusion] This study provided an effective method for the industrial production of high-active CAT-POD, and a reference for the expression of protein that contains prosthetic group.

Abstract:[Objective] To study the immunoregulatory effect of exopolysaccharide (EPS) from Lactobacillus plantarum JLK0142 on the RAW264.7 macrophages and immunosuppressed murine.[Methods] EPS were isolated and purified from the culture medium of L. plantarum JLK0142. The effects of EPS on proliferation, phagocytosis and nitric oxide (NO) secretion of RAW264.7 macrophages were determined by in vitro tests. Then the spleen index, proliferation of T lymphocyte and levels of IL-2 and TNF-α in serum of immunosuppressed mice induced by cyclophosphamide were determined by in vivo tests.[Results] EPS from L. plantarum JLK0142 could improve the proliferation activity of RAW264.7 macrophages in normal state at concentrations between 50 and 800 μg/mL, and significantly improve the phagocytic capacity of macrophages and the secretion of NO. Compared to control mice, the spleen index and T lymphocyte proliferation activity of the immunosuppressed murine were significantly increased in middle-does and high-dose EPS groups, while the levels of IL-2 and TNF-α in serum were also significantly increased in high-dose EPS group.[Conclusion] The EPS from L. plantarum JLK0142 can effectively improve the immune activity of RAW264.7 macrophages and antagonize cyclophosphamide induced immune suppression in mice.

Abstract:[Objective] We optimized carbon source for xylanase production by anaerobically halophilic alkaliphilic bacterium Alkalitalea saponilacus and characterized the enzyme.[Methods] Xylanase activity was determined by 2-nitro salicylic acid (DNS) method. The conditions for the extraction of crude xylanase were optimized and the enzyme was characterized.[Results] Xylanase activity by fermentation using 0.4% (W/V) sucrose+0.1% (W/V) birch xylan as carbon source was 3.2 folds higher than that with single substrate of birch xylan or sucrose. The maximal xylanase activity reached 590 IU/mg under the conditions of salinity between 2% and 6%, pH 7.0 and 55℃. Enzyme activity was significantly inhibited with Cu²⁺, Fe³⁺and Ni²⁺.[Conclusion] Xylanase produced by A. saponilacus can have potential for industrial production.

Abstract:[Objective] This work aimed to uncover the inhibitory effects of Bacillus velezensis L-1 on pear gray and blue mold, the inhibitory stability of its cell-free supernatant, and understand the molecular mechanism underlying the biocontrol processes.[Methods] We analyzed the inhibitory effects of strain L-1 on pear gray and blue mold both in vitro and in vivo, and observed the influences of strain L-1 on the mycelium growth of pathogens by microscopes. We determined the inhibitory stability of strain L-1 on Botrytis cinerea by oxford cup method and performed the complete genome sequencing of strain L-1 by Pacbio RSⅡ platform. We also annotated the obtained sequences through different protein databases to predict the mechanisms involved in the biocontrol processes.[Results] The control efficacy of strain L-1 against pear gray and blue mold was 92.88% and 77.47% respectively, and strain L-1 caused the swallow and abnormal growth of the pathogens' mycelium. Strain L-1 could grow normally in the broth containing 10% (W/V) NaCl, and its cell-free supernatant showed stable inhibitory effects against pear gray mold under acid, alkali, UV light, heat and protease treatments. Complete genome sequence analysis showed that there were 112 genes involved in the metabolism of different carbons, indicating its ability to grow on different carbon sources. Strain L-1 contained genes encoding the alimine and trehalose, that are related to stress resistance. Strain L-1 contained gene clusters related to the biosynthesis of a variety of polypeptide and polyketide compounds, such as surfactin, fengycin, bacillibactin, bacillaene, macolactin, difficidin, and bacilysin, and the genes encoding enzymes like β-1,3-glucase and chitinase, that could hydrolyze the pathogen cell wall. In addition, strain L-1 contained genes related to the biosynthesis of actoin, which could induce host resistance.[Conclusion] B. velezensis L-1 has the potential as an effective biocontrol agent.

Abstract:[Objective] We studied the effects of three biological agents on intestinal microbiota and immunity of white shrimp (Litopenaeus vannamei) in a water-recirculating cultivation system.[Methods] The composition of three biological agents were Bacillus subtilis, Candida utilis, nitrifying bacteria and photosynthetic bacteria (treatment 1); Proteinase, Acidophilus spp., Bacillus subtilis and Faecalis spp. (treatment 2); and Protease, yeast, Bacillus subtilis and Lactobacillus acidophilus (treatment 3). After feeding 30 days, the total numbers of bacteria and Vibrio in the intestine of L. vannamei were calculated using plate counting. The high throughput sequencing of 16S rDNA gene's V3+V4 region was used to determine diversity and composition of bacterial communities. Real time fluorescence quantitative PCR was used to analyze the expression levels of Toll-like receptor 1 (TLR1) and Dorsal.[Results] After the application of biological agents, the total number of bacteria in the intestine of L. vannamei was reduced to 7.22 CFU/g, and the growth of Vibrio was inhibited (5.01 CFU/g) in treatment 3. In treatment 3, the percentage of high sequences and Operational Taxonomic Unit (OTU) were increased to 98.8% and 326, Chao1, Simpson, Shannon was 581.34, 0.94, 5.52 respectively, which displayed the changes of bacterium abundance and diversity in intestine. Furthermore, the community structure of intestinal microbiota was optimized. Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, Firmicutes, Candidatus Saccharibacteria and Cyanobacteria were the main dominant phyla. However, the proportion of Bacteroidetes was significantly increased (40.7%) in the treatment 1 (P<0.05). The different expression levels of Toll-like receptor 1 and Dorsal were changed in different probiotics. In treatment 3, the expression level of Toll-like receptor 1 (TLR1) was 3.2 (P<0.05), which was 220% higher than control, meanwhile, the expression level of Dorsal was 4.7 (P<0.05), which was 370% higher than control.[Conclusion] Our study revealed that biological agents played an active role in the recirculating system, which could optimize the structure of intestinal microbiota and improve the expression levels of immune genes. In this case, these biological agents could serve as candidates for diseases prevention and healthy aquaculture. And the treatment 3 was the better.

Abstract:[Objective] Soil environmental problems caused by irrational fertilization are gradually becoming one of the important factors that restrict the sustainable development of agriculture in China. Soil fungi is a kind of important microorganisms in soil. Hence, it is significant to study the diversity and community distribution patterns of soil fungi under long-term fertilization and investigate the physical and chemical factors on the fungal community structure.[Methods] The black soil under the long-term fertilization experiment (1984-2017) in the northeast of China was adopted to be analyzed by routine analysis and Illumina MiSeq high-throughput sequencing. And then, the effects of long-term fertilization on soil nutrient contents and fungal community structures in black soil corn fields were studied.[Results] The results showed that the long-term application of nitrogen fertilizer significantly decreased pH in soil, but increased the yield of maize. The combined application of straw and fertilizer can increase the content of soil organic matter and total nitrogen. The results of operational taxonomic unites (OTU) curves showed that the long-term fertilization reduced the abundance and evenness of soil fungal sequences, and there was the lowest sequence number in the combined application of straw and fertilizer. Among the dominant communities, it has been found a total of five known fungal Phylum, Ascomycota, Basidiomycota, Zygomycota, Glomeromycota, Chytridiomycota. Among of them, Ascomycota accounted for not only 57.0% in the average of the total sequence, but also 70.35% in the soils with Fertilizer add high amount of straw treatment (NPK+S0.5). In the analysis of species abundance in soil fungal genus level, it has detected 109 known fungi. Humicola, Fusarium, Verticillium and Mortierella were the dominant genus. In addition to that, it was found that the abundance of Chaetomium, Trichocladium, Podospora and Preussia were high in the combined application of straw and fertilizer, and it all belong to a branch cluster. According to the analysis of diversity index, it can suggest that the combined application of straw and fertilizer can increase species abundance and community diversity. According to the Heatmap analysis, it can indicate that there were obviously different in the fungal community between the nitrogenous and non-nitrogenous fertilizers. In Redundancy analysis (RDA), the soil physical and chemical properties affected the community structure of soil fungi, especially soil pH, total nitrogen, total phosphorus and total potassium (TN, TP, TK), available phosphorus and available potassium (AP, AK) and ammonium nitrogen (NH₄⁺-N) is an important environmental factor.[Conclusion] The application of nitrogen fertilizer increased the yield. however, it caused soil acidification. Meanwhile, the application of nitrogen fertilizer increase the number of fungi, but the richness and diversity of fungi was decreased. Nevertheless, the combined application of straw and fertilizer can maintain soil healthy ecological environment and fungal community diversity.

Abstract:[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester, followed by using immobilized cells to catalyze the racemic ester.[Methods] We used rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester as esterase induction in the medium inoculated with soils to enrich the target microorganism. The strain with high activity for enantioselective hydrolysis of rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester was identified by 16S rRNA sequence analysis, morphological observation, physiological and biochemical properties. We optimized the immobilization conditions of the strain, studied the catalytic properties and operated stability of immobilized cells.[Results] We isolated a gram-negative bacterial strain which was identified as Methylopila. The optimal immobilization conditions were shown as follows:0.15% (V/V) of polymine, 0.2% (V/V) of glutaraldehyde, 6 g/L diatomite and 100 g/L cells. Compared with free cells, the optimum pH of immobilized cells changed from 8.0 to 8.5, the optimum temperature changed from 35℃ to 40℃, the stability of pH and temperature was improved. Cu²⁺, Mn²⁺ and Ca²⁺ can improve enzyme activity, but Zn²⁺ and Fe²⁺ inhibited it. The organic solvent tolerance of immobilized cells was higher than that of free cells. Affinity of immobilized cells with substrates decreased with the increased K_m value. We used the immobilized cells to catalyze rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester at the concentration of 200 g/L for 20 h, the remaining substrate was (S)-ester with ee_s>99.4% and the yield reached 47.8%. The relative activity of the immobilized cells remained 80% after 12 batches.[Conclusion] We established a process to produce (S)-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester from rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester by Methylopila sp. cxzy-L013 immobilized cells as the catalyst, which has a great potential for industrialization.