Abstract:Abstract:As cell factories，lactic acid bacteria are widely used in food，agriculture，pharmaceutical and other industries．Acid stress is one the important survival challenges encountered by lactic acid bacteria both in fermentation process and in the gastrointestinal tract．Recently，the development of systems biology and metabolic engineering brings unprecedented opportunity for further elucidating the acid tolerance mechanisms and improving the acid stress resistance of lactic acid bacteria． This review addresses physiological mechanisms of lactic acid bacteria during acid stress．Moreover，strategies to improve the acid stress resistance of lactic acid were proposed．

Abstract:Abstract:In nature，many insects，especially sap-feeding insects，harbor nutritional bacterial symbionts，which are called obligate endosymbionts．These bacteria co-evolved with their hosts for millions of years．Obligate endosymbionts are commonly found in specialized organs，named bacteriomes or mycetomes that consist of a number of insect’s cells (bacteriocytes or mycetocytes)．Obligate endosymbionts strictly maternally inherited，providing essential amino acids to the hosts，and relating to survival，reproduction and evolution of the insects．Because of enriched nutritional environment，compared to those free-living bacteria，the genomes of obligate endosymbionts have different characteristics，such as genome size，GC content，and gene deletion．Although the genomes of many insect endosymbionts have been carefully analysis，the gene functions of endosymbionts and the interactions between endosymbionts/hosts and endosymbionts remain unknown．Thus，to provide an insight into the co-evolution of endosymbionts and their hosts，further studies of endosymbionts at genetic level are required．

Abstract:Abstract:［Objective］The effect of flhDC，fliA，fliD and fliE genes involved in moving of Escherichia coli (E.coli) on the motility of lysogened strain by Stx2-encoding phage ΦMin27 was explored by gene knockout and phage lysogenic conversion．［Methods］ Using the lambda Ｒed recombinase system，the mutant strains of E.coli MG1655 named MG1655 △flhDC，MG1655 △fliA，MG1655 △fliD and MG1655 △fliE were constructed． Then the corresponding complemented strains by ligating amplified targeted genes into the low copy vector pUC18 at the BamHI and HindⅢ sites and transforming these plasmids into mutant strains were acquired． By lysogenic infection of Stx2-encoding phage ФMin27，the lysogens for mutants named MG1655△flhDCФMin27，MG1655△fliAФMin27，MG1655△fliDФMin27 and MG1655△fliEФMin27 were achieved． Subsequently，the motility of wild strain，the mutants，the complemented strains and the lysogens were detected． The changes of expression of the other genes involved in motility between wild strain and the lysogens before and after flhDC deletion by qＲT-PCＲ were analyzed．［Results］Lysogenic infection of Stx2-encoding phage ФMin27 could promote the expression of fliA and fliD gene and enhance the motility of MG1655．For flhDC deletion，higher expression of fliA and fliD gene of MG1655 appeared，but the motility had no change．However，lysogen for MG1655△flhDC lost the swimming motility．By gene transcriptional level detection，the expression of fliA and fliD gene of MG1655 △flhDCФMin27 was down-regulated significantly compared with MG1655 △flhDC，and no marked variation was observed for fliE gene． The single deletion of fliA，fliD and fliE gene had no effect on the motility of E.coli MG1655 and lysogened strain by Stx2-encoding phage ФMin27.［Conclusion］The results show that fliA and fliD gene together participated the regulation for flagella motility and flhDC gene could affect the motility of the lysogened strain by phage．It provides the theoretical basis for further research on the mutual regulation between phage lysogenization and host genes.

Abstract:Abstract:［Objective］To study the effect of ultra high pressure (UHP) on the cell membrane of Listeria monocytogenes．［Methods］We treated L． monocytogenes with different hydra high pressure from 100 to 500 MPa at 25℃ for 15 min．Then we determined the rate of cell inactivation by viable cell counts．We compared the morphological changes of treated and untreated cells using transmission electron microscopy (TEM)．We evaluated the membrane permeability by release of potassium ion (K+) or magnesium ion (Mg2+)，UV-absorbing substances and uptake of the fluorescent dye propidium iodide (PI)．We measured these parameters by spectrofluorometry，UV-spectrophotometer and atomic absorption spectrophotometer (AAS)．We used Na+/K+ -ATPase kit to measure the activity of Na+/K+ -ATPase.［Results］The pressure treatment at 300，350 and 400 MPa reduced the population of the bacteria from 9.00 to 5.20，3.27 and 1.35 Log CFU respectively，and no viable cells could be detected at 450 MPa．The structure changes revealed by TEM show that the cell membrane was damaged，the cell wall was breached and the cytoplasm aggregated and a large electron transmission area appeared which bring about the release of UV-absorbing substances，K+ and Mg2+ ions. Besides，the activity of Na+/K+ -ATPase was also decreased by UHP.［Conclusion］UHP could kill L．monocytogenes．

Abstract:Abstract:［Objective］We isolated actinomyces from the termitarium and studied its metabolites to find the antimicrobial compounds.［Methods］We determined the taxonomic status of target strain BYC 01 by morphological observation and 16s rＲNA sequence analysis. Growth rate method and agar disc diffusion assays were used to test the antimicrobial activities． Fermentation product was isolated and purified by various chromatographic methods，and the structure was determined by mass spectrum and nuclear magnetic resonance analyses.［Results］BYC 01 was identified as Streptomyces violaceoruber. The main antimicrobial ingredients of BYC 01 fermentation broth consisted in the ethyl acetate fraction of moderate polar part. The ethyl acetate extract of BYC 01 had strong antifungal activities against Valsa mali with inhibition rate of more than 90%，and activities against Ｒhizoctonia solani and Dothiorella gregaria with inhibition rate of more than 60% under the concentration of 100 μg/mL. Furthermore，the extract showed the intermediate antimicrobial activities against Candida albicans，Staphyloccocus aureus，Escherichia coli，Bacillus subtilis and Xanthomonas oryzae with the mean halo diameters ranging from 11.3 to 16.5 mm under the concentration of 30 μg/filter paper. A monomer compound was purified from the fermentation products，and was identified as fogacin on the basis of mass spectrum and nuclear magnetic resonance analyses. The compound fogacin and the positive control had similar antimicrobial activities against C. albicans with inhibition zone of 19.3 mm and 20.1 mm under the concentration of 30 μg/filter paper. ［Conclusion］Strain BYC 01 could be potentially developed as a new antimicrobial agent.

Abstract:Abstract: ［Objective］ This study aimed to screen endophytic bacteria with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and test the capability of growth promotion to its host.［Methods］In total 120 endophytic bacterial strains isolated from Panax ginseng were screened for 1-aminocyclopropane-1-carboxylate deaminase activity using the qualitative and quantitative methods． The obtained strain was also tested for its ability of nitrogen fixation using the Ashby agar plates and the gene of nifH，for its ability of phosphate solubilization using the Pikovaskaia’s plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry，for its ability of producing siderophores using the method of Chrome azurol S detecting，and its effect on promoting growth of Panax ginseng by laboratory and field experiments． The bacterial strain with ACC deaminase was identified based on morphology，physiological and biochemical traits，and 16S rＲNA sequence analysis.［Ｒesults］The bacterial stain JJ8-3 with the ability of producing ACC deaminase activity was obtained through screening，which its ACC deaminase activity was α-ketobutyric acid 6.7 μmol/(mg·h)．Strain JJ8-3 had other traits of phosphate solubilizing，nitrogen fixation，producing siderophores，and the ability of promoting growth of Panax ginseng． Strain JJ8-3 was identified as Pseudomonas fluorescens． ［Conclusions］ Strain JJ8-3 of endophytic bacterium with ACC deaminase activity from Panax ginseng was obtained and would lay the foundation for its further study and application on plant growth promotion．

Abstract:Abstract:［Objective］To isolate polyketide synthase (PKS) gene from medicinal Usnea longissima lichen forming fungi，and identify the function of obtained PKS.［Methods］We used Usnea． longissima lichen forming fungi to isolate PKS gene by nested PCR using degenerate primers and screening a Fosimid genomic library．MEGA 4.0. 2 program was used for phylogenetic analysis and RT-PCR was used to detect gene expression.［Results］ We obtained a gene cluster including non-reducing PKS (UlPKS5)，putative β-lactamase and putative dehydratase from Usnea longissima lichen forming fungi．UlPKS5 contained ketosynthase (KS)，acyl transferase (AT)，product template (PT) and acyl carrier protein (ACP) domain． Phylogenetic analysis shows that UlPKS5 belonged to non-reducing PKS group V，which involved anthraquinone biosynthesis.RT-PCＲ analyses reveal that the expression of UlPKS5 was up-regulated by sucrose (2% and 10%) and sorbitol (10%).［Conclusion］PKS(UlPKS5)，putative β-lactamase and putative dehydratase were related with anthraquinone biosynthesis in U. longissima.

Abstract:Abstract:［Objective］ We screened bacterial strains that have strong antagonism against Beauveria bassiana，an important pathogen of silkworm industry，and detected the antagonistic activity of lipopeptide metabolites.［Methods］We identified bacterium SWB16 by morphological observation，physiological and biochemical experiments，16SrＲNA，and gyrA gene sequence analysis，tested antagonistic activity of strain SWB16 against Beauveria bassiana by measuring the inhibition zone diameter using filter paper diffusion method (Kirby-Bauer method)，obtained lipopeptide metabolites of the strain using methanol extraction and observed the antagonism of strain SWB16 lipopeptide extracts against the conidia and hyphae of Beauveria bassiana，detected main ingredients and genes of lipopeptide metabolites by high-performance liquid chromatography-mass spectrometry and PCＲ amplification.［Results］ SWB16 isolated from tissue of plant Dioscorea zingiberensis C.H. Wright belongs to Bacillus amyloliquefaciens and showed high antagonistic activity to Beauveria bassiana，and the lipopeptide extracts of isolate SWB16 exhibited significant inhibition to conidial germination and mycelial growth of Beauveria bassiana．The result of mass spectrometric detection indicated main component of the lipopeptide metabolites were fengcin and iturin，and genes fenB，ituA involved in the synthesis of them were amplified in the genome.［Conclusion］ Bacillus amyloliquefaciens strain SWB16 could produce lipopeptide antibiotics with strong antagonism to the entomopathogenic fungus Beauveria bassiana，and the results suggested that strain SWB16 has potential application value for controlling white muscardine of economic insects including silkworm．

Abstract:Abstract:［Objective］Ectomycorrhizal fungi (ECMF)，important components in forest ecosystems，could form symbionts with wooden plant roots and participate in nutrient absorption.［Methods］Boletnus sp.(Bo 07)，Lactarius delicious (Ld 03) and Pisolithus tinctorius (Pt 715) isolated from Southwest China and Cenococcum geophilum (Cg 04) from Daqing Mountain，Inn Mongolia，China，were cultured in liquid Pachlewsk medium at 25 1℃ for 28 days with soil as sole K source．Fungal biomass，K uptake，efflux of protons and organic acids，and changes of soil K pools were measured to study K mobilization from soil by ECMFs.［Results］The fungal biomass，K concentration and uptake of Bo 07，Ld 03 and Pt 715 were much higher than Cg 04，indicating their strong abilities to absorb K and to adapt low K environment by bio-evolution and selection．K concentrations in culture solution were increased by ECMFs compared to blank control (without ECMF)．ECMFs could promote K release from the soil into culture solution． Bo 07，Ld 03 and Pt 715 increased significantly exchangeable K in soils，while structural K in soil was decreased by Bo 07 and Ld 03．They could thus mobilize unavailable K from ［Conclusion］ECMF isolates could mobilize unavailable K in soils．

Abstract:Abstract:［Objective］To evaluate the potential of Trichoderma longibrachiatum spore suspension against Heterodera avenae.［Methods］The parasitic and lethal effects of T．longibrachiatum spore suspension against the cysts of H．avenae were studied in vitro and observed under microscope．［Results］Microscopic observation showed that the spore suspension of T．longibrachiatum parasitized on the cyst surface，germinated a large number of hyphae，and grew on the surface of the cyst at the initial stage．Later，the cysts were completely surrounded by dense mycelium，and the contents of digestion in cysts was lysed，even some cysts produced vacuoles，and some were split up and finally the cyst was dissolved by the metabolite of T．longibrachiatum． In vitro studies showed that high concentrations of T．longibrachiatum spores had strong parasitic and lethal effects on the cysts of H．avenae，and the probable mechanism of parasitic and lethal effects of T．longibrachiatum against H． avenae were mainly by inducing and increasing chitinase，glucanase and caseinase activity．The cysts were parasitized by 93.3% at 18 days，the hatching of cysts were inhibited by 93.6% at 10 days when treated with the concentrations (1.5×108 CFU/mL) of T．longibrachiatum．［Conclusion］Trichoderma longibrachiatum had strong parasitic and lethal effects on the cysts of H．avenae，and has the potential as a new biocontrol agent．

Abstract:Abstract:［Objective］Chestnut blight fungus Cryphonectria parasitica and hypovirus constitute a model system to study fungal pathogenesis and host-virus interaction． Proteomic analysis of chestnut blight fungus upon hypovirus infection was conducted to find the differentially expressed host proteins．［Methods］According to the characteristics of this filamentous fungus，an optimized extraction protocol for fungal total protein was developed． Two-dimensional electrophoresis (2-DE) was used for comparative proteomic analysis of wild strain EP155 and hypovirus-infected strain EP713．The quantitative RT-PCR was applied to analyze mRNA expression level of protein-coding genes.［Results］In total 71 protein spots were detected to be differentially expressed on the base of EP155 of which 19 up-regulated and 52 down-regulated. Fifty-eight unique proteins were identified by mass spectrometry． Further study on quantitative RT-PCR indicated that the regulation of related host genes by hypovirus occurred at different levels.［Conclusion］ The TCA cycle of C．parasitica was weakened after hypovirus infection and the process of methylation was regulated by hypovirus． Meanwhile，viral regulation of virulence factors also contributed to the phenomenon of hypovirulence．

Abstract:Abstract:［Objective］In order to establish the vaccine against the contagious ecthyma，we constructed and characterized recombinant goatpox virus expressing F1L protein of Orf virus.［Methods］The F1L gene was amplified and cloned into the vector pUC-TK12 carrying the LacZ gene and a bidirectional promoter． With the help of lipidosome，the recombinant plasmid pTL-F1L was transfected into the BHK-21 cells，which had been infected by Gpv． The aim is to make the Gpv and pTL-F1L recombined randomly and get the recombinant virus，which was defined as rGpv-F1L. The rGpv-F1L was screened by blue plaque，and then the F1L recombination and translation were identified by PCR， indirect immunofluorescence and Western blot．By the means of TCID50，we evaluated the physicochemical properties of rGpv-F1L．Female mice were immunized with the rGpv-F1L，and the specific antibodies levels in serum were detected by ELISA.［Results］We obtained rGpv-F1L，which was stably expressing F1L protein．The results of biological characteristics showed the rGpv-F1L was sensitive to acids，alkalis，organic solvents and ultraviolet．The activity of specific antibodies significantly increased in mice infected by rGpv-F1L more than Gpv (P＜0.01).［Conclusion］In this research，we have successfully obtained the candidate vaccine，which is stably expressing F1L of Orf virus．Thereby the candidate vaccine with excellent antigenicity and biological activity provides new avenues for the prevention of contagious ecthyma and capripox．

Abstract:Abstract:［Objective］In order to use cellulosic biomass effectively，we screened a strain that can convert levoglucosan efficiently to carotenoids． ［Methods］Strain ZS1 was isolated from soil using levoglucosan of cellulosis pyrolysis products as sole carbon source．It was identified based on rDNA ITS sequence analysis and morphological characteristics．Phylogenetic tree was constructed to determine its taxonomic status．The conversion rate of levoglucosan was analyzed by high performance liquid chromatography．Carotenoid in fermentation liquid was detected by spectrophotometry.［Results］ The results show that ZS1could ferment levoglucosan efficiently and the consumption rate of levoglucosan reached 67.0% after 4 days．Strain ZS1 was identified as Rhodosporidium kratochvilovae (CGMCC NO:6365) based on the morphology features and ITS sequence analysis． After optimization of culture conditions，the conversion rate of levoglucosan reached 98.7% after 5 days．The content of carotenoid in cells was 427.1 μg/g (cell dry weight)．After conversion，460. 4μg/g (levoglucosan) of carotenoids could be produced with levoglucosan as carbon source.［Conclusion］The strain capable fermenting levoglucosan was identified as R．kratochvilovae that could produce carotenoids with levoglucosan as carbon source．

Abstract:Abstract:［Objective］To clone the full-length cDNAs of two laccase genes，vv-lac1and vv-lac6，from Volvaria volvacea，verify their encoded proteins with laccase activity and develop a heterologous expression and protein purification system for V．volvacea laccase genes.［Methods］The full-length cDNAs were cloned with rapid amplification of cDNA ends (RACE) technology and carried out in silico analysis．After modified by removing the sequence encoding signal peptide and adding the sequence encoding His-tag at 3' ends，the cDNAs were cloned into pPIC9K vector．The resulting constructs were transformed into Pichia pastoris GS115 for heterologous expression．The recombinant proteins were purified with Ni columns and the laccase activity were detected with ABTS assay.［Results］The full-length cDNAs of vv-lac1 and vv-lac6 are 1，599 bp and 1，554 bp，and contain19 and 15 exons，respectively．The predicted molecular weights of the proteins encoded by vv-lac1 and vv-lac6 are 57.3 kDa and 56.3 kDa，respectively．The predicted isoelectric points are 4.73 and 5.62，respectively．Both proteins are extracellular．The recombinant proteins RBvvlac1and ＲBvvlac6 are 70kDa，which may be modified by posttranslational modification．The solutions of the two recombinant proteins eluted by 150 mmol/L imidazole eluent have the highest laccase activity levels (333.17 U/L and 227.63 U/L).［Conclusion］The proteins encoded by the laccase genes vv-lac1and vv-lac6 from V．volvacea have laccase activity，the heterologous expression and protein purification system developed in this study is suitable for future studies of other laccase genes from V．volvacea or other fungi．