Abstract:Abstract:Thermoactinomycetaceae is a new and old prokaryotic group．The present taxonomic status is: Domain Bacteria，Phylum Firmicutes，Class Bacilli，Order Bacillales，Family Thermoactinomycetaceae． Study on this family is going on more than a century． Until now，this family comprises of 14 genera and 25 species． Members of the family Thermoactinomycetaceae are widely distributed in the terrestrial hot springs，high-temperature Daqu (a starter culture for Chinese alcohol fermentation)，compost，straw，bagasse and other places of high temperature，and marine sediments．The drug-resistance spores can survive in soil，water for long time． They also have great potential in both industrial and drug development，So they got a wide attention by scholars．This paper reviews the research progress of taxonomical studies and ecological diversity of the family Thermoactinomycetaceae，also introduces their potential applications in drug development and industrial production.

Abstract:Abstract:Vibrioparahaemolyticus is an important foodborne pathogen，of which the O3:K6 serotype caused many outbreaks in different countries since 1996．Based on the 10 years data (1992-2001) from China，gastroenteritis caused by Vibrio parahaemolyticus accounted for 31. 1% of foodborne disease outbreaks that were resulted from microorganisms．Most environmental strains ofVibrioparahaemolyticus are non-pathogenic strains．However，clinical strains can producethermostable direct hemolysin (TDH)，TDH-related hemolysin，and other virulence factors．Here we reviewed three commonly used molecular markers forVibrioparahaemolyticus，including species-specific genes，the virulence genesand pandemic group-specific genes，so that to provide references for the rapid detection of Vibrio parahaemolyticus and the identification of its pathogenic factor.

Abstract:Abstract:［Objective］Lager brewing yeasts (Saccharomyces pastorianus)，the natural hybrids of S．cerevisiae and S．eubayanus，are usually heterothallic polyploidy or aneuploidy．Their intricate ploidy is a great challenge to genetic studies and strain improvement． Haploid breeding is an effective method to overcome these difficulties．Also，haploid strains play an important role in scientific research and breeding． However，lager brewing yeasts only divide asexually and hardly bear spores under normal conditions，so it is very difficult to get haploid strains from them．In this study，we established comprehensive methods to induce，separate and identify haploid strains of industrial brewer's yeast．［Methods］First，we selected efficient sporulation medium to induce the sporulation of an industrial brewer's yeast strain G-03，and then isolated spores from vegetative cells and formed colonies on YPD plates． After that，flow cytometry was used to determine the ploidy types of the pre-judged haploid candidates．Ultimately，we analyzed the genotypes of the segregants by PCR reaction and mating test in order to get precise results．［Results］Using this protocol，we obtained 26 yeast segregants by spore isolation，and 4 of them pre-judged as haploid candidates were finally confirmed as haploid by flow cytometric analysis．Two of them were MATa and others were MATα．By scanning electron microscope (SEM)，the cells of 4 haploid segregants showed similar morphology to each other but had obvious differences compared with the parent strain．Pseudohyphal growth occurred in parent cells after long-period cultivation but none was found in haploid segregants．［Conclusion］Sporulation of industrial brewer's yeast and germination of their spores was difficult but not impossible．Nevertheless，the screening and identification of haploid segregants were more challenging.

Abstract:Abstract:［Objective］ To find anti-phytopathogen compounds from endophytic fungi associated with the endangered species Heptacodium miconioides．［Methods］Fungi from H．miconioides with antifungal activities were isolated according to the plate growth inhibition method． The fungus with preferable antifungal activities was identified by morphological identification and 5. 8S rＲNA sequence analysis．The bioactive metabolites were isolated and purified by chromatographic methods; the structures were determined by spectroscopic analysis．［Results］ Alternaria solani QZH 10 showed better antifungal activity against Ｒhizoctorzia solani and Valsa mali with the inhibition rates of 89.1% and 67.9%，respectively. The ethyl acetate crude extract of QZH 10 had strong antifungal activity against Magnaporthe oryzae with the rate of 100. 0% under the concentration of 100 μg/mL. Two antifungal metabolites altersolanol A and 6-O-methylalaternin were isolated and determined from QZH 10．Altersolanol A possessed strong activity against M．oryzae with the inhibition rate of more than 85%，6-O-methylalaternin had the mightily activity against V．mali with the inhibition rate of 100.0% under the concentration of 100 μg/mL．［Conclusion］Altersolanol A and 6-O-methylalaternin are potential fungicides originated from microorganisms.

Abstract:Abstract: The alkaliphilichemicellulolytic bacterium Bacillus sp．N16-5 was isolated from Lake Wudunao in Inner Mongolia．It has a broad substrate spectrum and exhibits a capacity to utilize complex carbohydrates such as galactomannan，xylan and pectin．Previous transcriptional analysis of differential carbohydrate utilization by Bacillus sp．N16-5 has identified a putative gene cluster related to xylan utilization．It contains a putative xylo-oligosaccharide ATPbinding cassette (ABC) transporter encoded by xynEFG gene cluster．［Objective］xynE gene is predicted to encode an extracellular solute-binding protein of the ABC transporter．Here，the physiological roles of xynE on the xylan utilization was investigated by gene deletion．［Methods］We obtained the xynE deficient strain N16-5(ΔxynE) through homologous recombination using a temperature sensitive shuttle vector pNNB194． The effects of xynE on xylan utilization by N16-5 were detected by comparing the growth profiles on xylan of the wild type and mutant strains as well as the variation of reducing sugars concentration in the medium during cultivation．We further verified the phenotype by constructing the complementary strain．Moreover，the substrate specificity of XynE was illustrated by the HPLC analysis results of the xylan medium components，which was supplemented with the growth profiles of the wild type strain and N16-5 (ΔxynE) strain on xylose．［Results］ Compared with the wild type strain，strain N16-5 (ΔxynE ) had a delayed exponential phase， obtained a lower maximum optical intensity OD600 value，and presented the accumulation and depletion of reducing sugars during cultivation．The complementary strain retrieved the phenotype of wild type strain，and grown slightly better than it．HPLC analysis showed that N16-5 ( ΔxynE ) strain degraded the xylan substrates more slowly than wild type，xylooligosaccharides like xylotetraose，xylotriose and xylobiose began to accumulate after 16 h cultivation．Moreover it still maitained a large number of the degradation product xylobiose in the medium after 60 h． When cultured in xylose medium，strain N16-5(ΔxynE) performed similar growth profile with the wild type strain． ［Conclusion］XynE played an important role in rapidly and effectively utilizing xylan in Bacillus sp．N16-5 and specifically related with xylo-oligosaccharide uptake．

Abstract:Abstract:［Objective］To reconstitute the in vitro catalytic activity of the individual dehydratase or cyclase domain of bifunctional bovicin HJ50 synthase BovM，and lay a foundation for the further investigation of catalytic mechanism of class II lantibiotic synthase LanM．［Method］The truncated proteins of BovM containing the N-terminal dehydratase domain or C-terminal cyclase domain were expressed in E．coli and purified． Substrate BovA，the precursor of bovicin HJ50，was incubated with these truncated BovM proteins in in vitro reaction system．The antimicrobial activity assay and MALDI-TOF MS analysis were used to monitor the dehydratase or cyclase activity of these truncated proteins． Meanwhile，the synergistic activities of both truncated proteins were tested in vivo and in vitro．［Results］The N-and C-terminal domains of BovM possessed dehydration and cyclization activity respectively．However，no synergistic activity was detected between these two functional domains．［Conclusion］The individual functional domains of BovM could execute their corresponding functions independently，but the intactness of BovM was important for its full modification activity.

Abstract:Abstract:［Objective］ We cloned a lipase gene，lipC24，from Burkholderia sp．ZYB002 and characterized the recombinant lipase LipC24．［Method］Based on the known genomic DNA sequence from Burkholderia cecapia JK321，we designed a pair of specific primers for the lipC24 gene and then obtained the full length of lipC24 gene．The lipC24 gene fragment enconding the mature peptide LipC24 was then subcloned into expression plasmid，pACYC-Duet-lipB，and expressed in E．coli．The recombinant protein，LipC24，was purified to homogeneity by HisTrap HP chromatography column and HiTrap DEAE FF chromatography column．［Results］We expressed the lipC24 gene from Burkholderia sp．ZYB002 in E．coli Origami 2(DE3) ．Nucleotide sequencing revealed that the lipC24 gene had an open reading frame of 1317 bp，and the deduced amino acid sequence of LipC24 corresponded to 438 amino acid residues，including a conserved -G-X1 -S-X2 -G-motif．The relative molecular weight of the purified LipC24 was about 45 kDa．The purified LipC24 displayed hydrolysis activity to various 4-nitrophenyl esters and substrate preference for the medium chain length 4-nitrophenyl-esters．The optimal temperature was 40℃ and the optimal pH was 7.5．The lipase was stable between pH 7.0 and 8.0 for 24 hours．However，the half-life was only 16 min at 40℃．［Conclusion］The LipC24 was a 45 kDa protein，a mesotherm and neutral lipase.

Abstract:Abstract:［Objective］ We studied the cloning and expression of lasB encoding solvent-resistant protease from Pseudomonas aeruginosa PT121．The recombinant protease was then characterized and applied in peptide synthesis．［Methods］The PCR primers were designed to acquire the open read frame (ORF) of lasB according to similar protease gene (pseudolysin) reported in the literature．Inducible expression plasmid pET22b-lasB was constructed and expressed in E.coli BL21 (DE3)．The recombinant protease was then characterized and applied in peptide synthesis．［Results］ The protease PT121 was defined as metalloproteinase M4 family according to sequence blast．Gene sequence analysis shows that lasB encodes signal peptide，pro-peptide and mature peptide．Mature protein contains 301 residues with molecular weight of 33 kDa．One-step preparation of the recombinant proteases PT121 was optimized by breaking cell wall．The specific activity of protease PT121 reached up to 7700U/mg，and it was stable similar with wild type PT121 from P．aeruginosa PT121 in temperature，pH and organic solvent．The synthesis rate of various dipeptides in 50% DMSO was effective，especially productivity of aspartame precursor reached up to 91%．［Conclusions］ Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.

Abstract:Abstract:［Objective］We studied the effect of methyl bromide fumigation on soil edaphic denitrification．［Methods］We adopted nosZ-PCR-RFLP (restriction fragment length polymorphism) method，nosZ-MPN-PCR (Most-Probable-Number-PCR) counting method and soil nitrate elimination rate method，to explore the effect of methyl bromide fumigation on community structure，quantity and activity of denitrifying bacteria in soil．［Result］After methyl bromide fumigating soil for 100 d，soil denitrification did not change obviously (P＞0.05)．Margalef index，Shannon-wiener index and Evenness index had no significant difference (P＞0.05) in nosZ denitrifying bacterial communities between fumigated soil and the control．There were Ｒhodopsendomonas，Pseudomonas fluorescens，Herbacspirillum，uncultured bacterium partial in both of them．However，Azospirillum，Rhizobium melibei，Nitrosospira multiformis were exclusively found in the control，and Uncultured Azospirillum sp，Mesorhizobium sp were in fumigated one．Moreover，the number of denitrifying bacteria in the control resolved by nosZ-MPN-PCR(Most-Probable-Number-PCR) was 1. 4 times higher than that of the fumigated one．［Conclusion］After 100 d fumigating soil，the composition of nosZ denitrifying microbial community and the population of denitrifying bacteria changed．Furthermore，there was no difference in denitrification between the fumigated soil and the control.

Abstract:Abstract:［Objective］ We screened and isolated bacterial strains from the perennial administration quinclorac paddy fields to degrade quinclorac．［Methods］ Strain MC-10，which can degrade quinclorac efficiently，was screened by enrichment and selective medium． The strain was identified by morphological，physio-biochemical characteristics and 16S rDNA gene sequence analysis．［Results］MC-10 was identified as Arthrobacter sp．Under the optimal growth conditions with inoculum concentration of 5% ，at 28 C，pH 7 for 7 d，MC-10 degraded more than 90% of quinclorac．MC-10 can effectively degrade quinclorac when quinclorac initial concentration at 1 mg/L－100 mg/L．And the strain MC-10 has a good ability to survive in the soil．More than 70% quinclorac in the soil can be degraded efficiently after 7 days of cultivation．［Conclusion］ Arthrobacter sp．MC-10 could be a promising microorganism in dealing with quinclorac pollution．

Abstract:Abstract:［Objective］To study the role of gspL gene in avian pathogenic Escherichia coli．［Methods］The gspL mutant of Avian pathogenic Escherichia coli (APEC) was constructed by homologous recombination assay．The growth characteristics，the ability of adhesion and invasion to DF1 cells，the virulence genes transcription level and median lethal dose (LD50) were analyzed between the gspL mutant strain and the wild strain．［Results］Compared with the wild strain，the mutant strain had no significant difference in the growth status．However，its ability of adhesion and invasion was significantly lower．The transcription of genes pfs，fyuA，iss and vat increased obviously，the tsh decreased and the transcription level of luxS，ibeA，stx2f and ompA had no significant change． LD50 showed that the gspL mutant strain had 12-fold increase in virulence．［Conclusion］The deletion of gspL gene could abate the ability of adhesion and invasion，regulate and control some virulence gene transcription level，enhance the virulence of APEC． The results show that the gspL gene play roles in pathogenicity of APEC．

Abstract:Abstract:［Objective］A mass mortality of tilapia broke out in an aquaculture farm in Panyu，Guangdong Province in May，2013．Affected fish showed blackening of body color，haemorrhageing on surface，scales shedding，fin rotting，and the presence of yellow liver，dark red spleen，enlarged gallbladder and ascitic fluid in the abdominal cavity．The purpose of this research was isolating and identifying the pathogen． ［Methods］We isolated a suspicious bacteria strain PYS1 from diseased fish with significant pathological signs．The homology of 16S rRNA gene sequence of strain PYS1 and its morphological，cultural，and physical and chemical characteristics were studied for its identification．Its pathogenicity was investigated by recursive infection experiment and histopathological study．Its effective medicines was screened by antibiotic sensitive test．［Results］The results showed that strain PYS1 was Plesiomonas shigelloides clustered with P．shigelloides strains isolated from other fishes in the molecular phylogenetic tree of 16S rRNA gene sequences．Strain PYS1 was multiple drug resistant and only sensitive to a small part of 31 tested antibiotics (e.g.，ceftriaxone，cefaclor，cefazolin，etc.)．The symptoms of tilapia (O. niloticus) artificially infected with strain PYS1 were similar with natural infected fish．The half lethal dose (LD50) of strain PYS1 to tilapia was 1.425×108 CFU per fish． Paraffin sections showed intestine，liver，spleen，kidney and heart tissue injury caused by the strain．［Conclusion］ Our study demonstrated that P. shigelloides was the pathogen of cultured tilapia in the aquaculture farm and first reported its bacterial pathogenicity on Nile tilapia.

Abstract:Abstract:［Objective］ To construct a 5-oxomilbemycin producing strain by knocking out milF gene in Streptomyces hygroscopicus HS023． ［Methods］Plasmid pMSST-ΔmilF was constructed and introduced into milbemycin industrial strain Streptomyces hygroscopicus HS023，and milF mutant F2-18 was selected by PCR amplification．［Results］Fermentation experiments showed that no milbemycins was produced in F2-18，but 5-ketomilbemycin，the intermediate of milbemycin，was obviously accumulated，and the fermentation titer was enhanced．［Conclusion］ Genetically engineered strain can simplify the synthesis of milbemycin oxime and lepimectin chemical from milbemycin．