Abstract:Abstract:We reviewed the methods for identification of microorganisms on the surface of historic stones，including nucleic acid analysis， cell membrane analysis， secondary metabolites analysis and the traditional culture analysis．After comprehensive comparison of the advantages and disadvantages of each method，we addressed the biological protection of stone artifacts． The establishment of rapid detection of microorganisms on the historic stones is important to prevent corrosion caused by microorganisms and to protect our valuable cultural heritage．

Abstract:Abstract:Spinosad is a novel macrolide insecticide produced by Saccharopolyspora spinasa，widely used in agriculture．Here its biosynthetic pathway，and key regulatory nodes were reviewed and analyzed．A synthetic strategy to reprogram spinosad biosynthetic pathway was proposed．

Abstract:Abstract:S-layer proteins are crystalline arrays composed of numerous identical protein or glycoprotein subunits． They comprise the outermost cell envelope component in most archaea and many bacteria． The remarkable properties of S-layer proteins are their capability to self-assemble and specific binding，which give S-layer proteins a wide application potential in nanotechnology and biomimetics． Recently most study in this field was concerned on the S-layer proteins of Bacillus species． This review provided a brief survey of the current state of the study about the genetics，function，properties，pathogenic correlation and application of the S-layer proteins of Bacillus species． The aim of this review was to gain more knowledge about the function of S-layers and lead to the development of new types of surface display system and recombinant vaccines，diagnostic agents and biocompatible surfaces．

Abstract:Abstract:［Objective］ This study characterized the phylogenetic diversity of soil bacteria producing nematode-attracting volatiles and their nematode-attracting compounds． Results would enhance our understanding on the interaction between nematodes and soil microorganisms and potentially enhance the biocontrol efficiency when combined the attractants with nemacides． ［Methods］Bacteria producing volatiles with functions of nematode-attracting activities were isolated from 187 agricultural soil samples collected in 26 provinces of China with the method of double-Petri dishes． The phylogenetic diversity of these bacteria was characterized by RFLP-16S rRNA gene sequence analysis． The nematode-attracting volatiles of bacteria were detected using the SPME-GC /MS，and volatile compounds with attractive activity were determined by confirming with individual commercial compounds． ［Results］ Among the 3800 bacteria isolated from the 187 soil samples，196 isolates(5. 16% of the total) showed attractive activity(AN)more than 30% to Panagrellus redivivus．Of the 196 isolates，66 (1.74% ) showed AN≥70%，62 isolates ( 1. 63%) showed AN between 50% and 70% ，and 68 isolates (1.79%) showed AN less than 50%．Phylogenetic analysis revealed that the 196 bacteria were clustered into 5 groups: Bacilli，Gammaproteobacteria，Alphaproteobacteria，Sphingobacteria and Actinobacteria． But，Bacillus were the dominant，which covered 13 species． And 11 volatiles with nematode-attracting activity were determined，including benzaldehyde，2-heptanone， benzyl benzoate， ethyl palmitate， (+) -longifolene， benzyl alcohol， p-anisaldehyde，vanatone，ethyl butyrate，isovanilin and d-alaninol． ［Conclusion］ Some species of bacteria in agriculture soil can produce volatiles to attract nematodes．

Abstract:Abstract:［Objective］ Actinomycetes (actinobacteria) are getting more and more recognized as a natural source for new drug exploration． In order to find new lead compounds，the diversity and selected bioactivities of cultured actinomycetes in the Baltic Sea (Germany) were investigated． ［Methods］One hundred sediment samples were collected from south of the Baltic Sea，of which 809 purified cultures of actinomycetes were obtained by using 7 media． The phylogenetic analysis of 280 selected strains based on 16S rRNA gene sequences was carried out． In addition，activities of 21 enzymes，which play a role in metabolic processes，and anti-microbial activities were determined． ［Results］ Fifteen genera and eight possible new species of actinobacteria were identified． Members of 3 genera were not isolated from marine habitats before． Of the 280 strains 21% and 20% inhibited the growth of Bacillus subtilis and Staphylococcus lentus，respectively． More than 75% of the strains exhibited 8 types of enzymatic activities，including esterase lipase (C8)，catalase and naphthol-AS-BIphosphohydrolase． ［Conclusion］ Baltic Sea provides a rich diversity of actinobacteria regarding the phylogenetic analysis and the biological activities． Research and utilization of marine actinomycetes should be strengthened．

Abstract:Abstract:［Objective］ A new method used to heterologously express ［FeFe］-hydrogenase in Escherichia coli was investigated in our present study． ［Methods］ By homologous recombination，three assistant genes (hydE、hydF and hydG) for hydrogenase were integrated into the chromosome of E． coli BW 25113-10，in which all hydrogenase genes were inactivated． A hydrogenase structural gene hydA from Clostridium butyricum was used to test the hydrogenase maturation ability of the recombined E． coli． BW 25113-13 ［Results］ The corrected integration of the three assistant genes was confirmed by PCR，and RT-PCR results indicated that the three accessory genes were transcripted in the recombinant． The active expression of hydA indicated that the constitutively expressed accessory proteins could assist the maturation of the ［FeFe］-hydrogenases． ［Conclusions］ A simplified ［FeFe］-hydrogenase expression recombinant E.coli BW25113-13 was constructed． It would lay foundations for the functional screening of ［FeFe］-hydrogenases and the construction of novel hydrogen producing pathways in E.coli．

Abstract:Abstract: ［Objective］In order to optimize precursor supply for L-arginine biosynthesis，we constructed a Corynebacterium crenatum 8-193 mutant with gamma-glutamyl kinase gene (proB) in-frame deletion．The effects of proB knock-out on physiological characteristics of the mutant were investigated．［Methods］ The upstream and downstream fragments of proB were cloned from C．crenatum 8-193 chromosome and ligated to integration vector． The mutant C．crenatum 8-193-ΔproB was obtained by homologous recombination．The mutant phenotype can be reversed by complementation with proB gene from the expression vector．The physiological characteristics of the mutant were investigated by measurement of the activities of phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PYC)．［Results］ The proB gene in-frame deletion was screened and confirmed by PCR，gamma-glutamyl kinase determination and complementation． The mutant lost the ability of growth on minimal medium without proline addition．The proB knock-out mutant resulted a decrease of cell mass by 9. 6% and an increase of L-arginine accumulation by 13. 6% compared with that of the parent strain． The analysis of by-products of fermentation broth showed that the concentrations of glutamate-related and aspartate-related amino acids increased，and the concentrations of α-ketoglutaric acid，PEP and succinic acid decreased． The specific activities of PEPCx and PYC increased in 8-193-ΔproB．［Conclusion］ The proB gene knock-out of the strain 8-193 blocked branch catabolism of L-glutamate and improved efficiency of the glucose utilization and L-arginine accumulation．

Abstract:Abstract:［Objective］ Paenibacillus mucilaginosus is widely used as microbial fertilizers，therefore，rapid identification of this species by Polymerase Chain Reaction (PCR) is necessary for detection and ecological evaluation of microbial fertilizer．［Methods］ A species-specific primer pair of P．mucilaginosus，orf06701-F (5'-ATGGAGGAAACATGGGGTGA-3')/orf06701-R (5'-TCAGGAATGAAGGCCCCCTT-3') was designed． Optimization of PCR，specificity and sensitivity determination were followed and the rapid identification was established．［Results］ A single band with 333 bp was consistently amplified from all the strains of P．mucilaginosus tested and negative results were obtained from all the reference strains，such as other strains of Paenibacillus and Bacillus． The PCR detection limitation was 400－1000 cells per assay，indicating the sensitivity of the rapid culture-PCR method．To verify the rapid identification，soil was sampled and cultured． Five of 11 soil isolates were rapidly identified as P． mucilaginosus by the method，and the similarity of 333bp sequences were 100%．［Conclusion］ The methods can be used in the rapid identification of P．mucilaginosus and the results will provide technical supports for the detection and ecological evaluation of microbial fertilizer．

Abstract:Abstract: ［Objective］ We developed the transformation methods of the strain Chaetomium globosum NK-102．［Methods］ We constructed plasmid pUCATPH-Pgap and compared the transformation efficiency with pUCATPH and pCM768． We established the PEG mediated protoplast transformation and Agrobacterium tumefaciens EHA105 mediated transformation methods． ［Results］In protoplast approach，approximately 3－5 transformants/μg DNA could be obtained．The highest efficiency of transformation was obtained by employing pUCATPH-Pgap．A．tumefaciens EHA105 successfully mediated T-DNA insertion into the genome of C． globosum NK-102 and the transformation rate was 3.2×102 transformants /107 spores． The transformants retained stable after generations． Southern blot analyses confirmed that the DNA had integrated into the chromosomal DNA of C． globosum NK-102．［Conclusion］The transformation systems were good basis for selection of C． globosum mutant strains that effectively utilizing cellulose．

Abstract:Abstract: ［Objective］ To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide． ［Methods］We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion，B．martensi Karsch(BmK ITa1) gene at the control of ie-1 promoter． The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells． Recombinant viruses were purified by the end point dilution and fluorescent spot purification．［Results］We successfully screened the recombinant SpltMNPV-Δegt-Pph-egfp-ie-1-BmK ITa1 of which the egt gene was knocked out and expressed the mature peptide of the BmK ITa1 gene at the control of ie-1 promoter． Bioassays showed that，compared to the wide-type SpltMNPV，the speed of the recombinant virus killing the S． litura (LT50) increased by 0.7－0.8 days．［Conclusion］ The insecticidal effect of SpltNPV could be increased by inserting the foreign gene，which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S． litura．

Abstract:Abstract:［Objective］We screened a thermophilic xylolytic bacterium that produced fuel ethanol from a high-temperature oil reservoir，and provided microbial resources to genetic engineering strains construction and consolidated bioprocessing．［Methods］We adopted Hungate anaerobic technique to isolate strain xyl-d from oil reservoir water sample enriched for two years from Shengli Oilfield in China，and we identified strain xyl-d with morphological，physiological，biochemical and phylogenetic analysis．［Results］ Strain xyl-d was gram-negative，rod-shaped，spore-forming and strictly anaerobic．The growth temperature ranged from 30℃ to 85℃ (optimum 65℃) and the pH ranged from 3.0 to 10.0 (optimum 7.5) and salt concentration was 0%－4%(optimum at 2.0%)．It converted D-xylose into ethanol，acetate，CO2，trace amount of iso-butanol and propionate． The genomic DNA G + C contents of strain xyl-d was 45. 6 mol% ． Based on 16S rRNA gene sequence，strain xyl-d was most close to Thermoanaerobacter wiegelii DSM10319T and Thermoanaerobacter ethanolicus DSM 2246T both with the 99. 3% similarity． It produced more ethanol and less acetate at initial pH 8. 5 than other pH． Ethanol yield was increased significantly with yeast extract，and ethanol became the main end product． In addition，growth of strain xyl-d was inhibited obviously with ethanol concentration more than 7% (V/V)．In the optimum growth conditions，xylose degradation rates reached to 91.37%．［Conclusion］ Strain xyl-d was thermophilic，high xylose conversion rate，acidotolerant anaerobe． It was a potential bacterium that can be used for consolidated bioprocessing．

Abstract:Abstract:［Objective］ To isolate and identify an alkaliphilic mannanase producing bacterium，purify and characterize mannanase thereof． ［Methods］ Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia，China． The morphological，biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15． Mannanase produced by strain HMTS15 was purified by four steps including (NH4) 2 SO4 precipitation，cellulose DEAE-sepharose，twice Superdex 200． The enzyme properties including optimal temperature，optimal pH，thermal stability，pH stability，NaCl tolerance，metal ion tolerance，EDTA and SDS tolerance were tested． ［Results］ Strain HMTS15 was Gram-positive rod．Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10℃ to 45℃．The G+C content of the DNA was 40 mol%．Phylogenetic analvses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus． The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE． The optimal catalytic activity was showed at 75 ℃ and pH 10． The mananase was stable up to 60℃ and retained about 60% residual activity at 65℃ for 30 min． The ions Fe2+，Mn2+，Co2+，Zn2+，Ag+，Hg2+and EDTA inhibited the acitivity of the mannanase．［Conclusion］ Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens． The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH．

Abstract:Abstract:［Objective］ We studied the antibiotic activity and selective cytotoxicity of β-1，3-1，4-glucanase from endophytic Bacillus subtilis SWB8． ［Methods］ Based on gel permeation chromatography， sodium dodecyl sulfatepolyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry methods，protein fragments of β-1，3-1，4-glucanase from endophytic Bacillus subtilis strain SWB8 were purified and identified． Then，β-1，3-1，4-glucanase was used to evaluate the antimicrobial activity against Staphylococcus aureus，Enterococcus faecalis，Bacillus subtilis，Escherichia coli，Salmonella typhi，Salmonella paratyphi A，Shigella dysenteriae，Candida albicans and Cryptococcus neoformans and cytotoxicity against human pulmonary adenocarcinoma cells (A549) and human bone marrow mesenchymal stem cells (MSCs) by using the disc diffusion，methyl thiazolyl tetrazolium and flow cytometry methods，respectively．［Results］ Bacterial β-1，3-1，4-glucanase showed broad antimicrobial spectrum against all nine bacterial and fungal strains． Furthermore，β-1，3-1，4-glucanase possessed significant anticancer activity against A549 cells that the IC50 and IC90 values were 11.5 and 20.1μg/mL，respectively． The percentage of apoptotic A549 cells treated with different concentrations of β-1，3-1，4-glucanase was significantly increased from 4. 43% of the control to 43.1% of 19.2μg/mL glucanase in a dose dependent manner． In contrast， these changes could not be observed in human bone marrow mesenchymal stem cells． ［Conclusion］β-1，3-1，4-glucanase could be a potential source of desirable antimicrobial agent，or anticancer compounds with higher efficiency and lower toxicity．

Abstract:Abstract:［Objective］We isolated and identified endophytic bacteria from halotolerant plants collected from coastal shoal of Nantong and investigated their heavy-metal tolerance and plant growth promoting potential．［Methods］ In total 45 strains were obtained from 4 halotolerant plants and 23 representative isolates were selected to detect their tolerance against NaCl and heavy metals of Cu2+，Pb2+，Cd2+，Zn2+，Hg2+; plant growth promoting index of nitrogen fixation，phosphate solubilization，indoleacetic acid (IAA) production and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase．[Results] Most of the isolates could grow under high consistency of Cu2 + and Pb2 + ． Of the bacteria 26. 1% had the ability of nitrogen fixation，21. 7% of phosphate solubilization，60． 9% of IAA production and 39. 1% of ACC deaminase activity． The results of 16S rRNA sequencing show that they belonged to the genera of Bacillus， Halobacillus，Oceanobacillus，Exiguobacterium，Serratia，Brevundimonas，Vibrio and Staphylococcus． Among them，strains KLBMP 2432 and KLBMP 2447 were potential novel species． ［Conclusion］ The halotolerant plants located in the area of coastal shoal contain a variety of endophytic bacteria as well as the source of novel taxa． Some of them had the ability of plant growth promoting and high resistance against heavy-metal Cu2 + and Pb2+．

Abstract:Abstract:［Objective］ The aim of this study is to identify a polycyclic aromatic hydrocarbons (PAHs)-degrading bacterium isolated from deep-sea hydrothermal environment， including its taxonomy， characteristics and mechanism involved in PAH degradation ［Methods］ The phylogeny was studied by 16S rRNA gene clone，and the degradation rates against different PAHs were determined by GC-MS． Meanwhile，PAH-degrading gene cluster was cloned by the genomic Fosmid library construction; the function of the key degrading-gene expression was examined by RT-PCR and qPCR to observe gene expression in the response to different PAHs． ［Results］A PAH-degrading strain TVG9-Ⅶ was isolated from the hydrothermal chimney sample of the Lau basin in Southwest Pacific Ocean．It showed 99. 7% similarities with 16S rRNA gene of Novosphingobium indicum strain H25T ． The degradation rates of this strain against phenanthrene，fluoranthene and pyrene were 95．2%，57.3% and 69. 6% in 21 days，respectively．A gene cluster，containing PAHs initial dioxygenase genes pheA1a and pheA1b，was obtained from genomic fosmid library，with the insertion size of 12.522 kb．The gene pheA1a was enhanced by 4. 2 folds in mRNA expression in presence of phenanthrene，but expression enhancement was not observed in other tested PAHs including naphthalene，pyrene and fluoranthene．［Conclusion］ Strain TVG9-Ⅶ is isolated from deep-sea hydrothermal environment in genus Novosphingobium． It can degrade many kinds of PAHs，especially the high-weight-molecular PAHs．

Abstract:Abstract:［Objective］ Listeria monocytogenes (Lm) is an important pathogen that can cause serious listeriosis in humans and animals． The pathogenicity of Lm has a close relationship with the PrfA protein regulating the expression of virulence genes． Therefore，we studied the regulation functions of PrfA and its role on Lm's virulence．［Methods］ The prfA genes of LM4，serotype 1/2a，and F4636，serotype 4b，were deleted by homologous recombination technology， and the biological characteristics of the mutants were further studied．［Results］ The prfA gene deleted strains LM4ΔprfA and F4636ΔprfA and their back mutation strains were successfully constructed． The results show that the hemolysis activity was lost in prfA deleted strains and was recovered in the reverse mutant strains． The prfA deleted strains lost phospholipase activity; their adhesion and invasion ability significantly decreased． Furthermore，their 50% lethal doses (LD50) were 5 logs higher comparing with wild type strains．［Conclusion］ PrfA regulates hly，plcB and inl gene family and affects significantly Lm's virulence．

Abstract:Abstract:［Objective］ Paracin 1.7 was a bacteriocin extracted from Lactobacillus paracasei HD1.7．In this study，we investigated quorum-sensing mechanism that regulates the metabolism of Paracin 1.7 in strain HD1.7．［Methods］ The antibacterial activity of strain HD1. 7 under different growth conditions was analyzed using an agar-well diffusion method，and the cell density was controlled by adjusting nutrient levels in the culture medium． ［Results］The antibacterial activity of strain HD1. 7 depended on cell density．Strain HD1.7 exhibited the antibacterial activity when the cell density reached a threshold (OD600 of 0.8 and cell dried weight of 0. 3311g/L)．Addition of different concentrations of a signaling molecule in the supernatant of the fermentation broth affected the antibacterial activity． After removing the signal molecule，the antibacterial activity was significantly decreased．［Conclusion］ Paracin1. 7 was the quorum-sensing signaling molecule in the HD1.7 fermentation broth and it could be auto-induced．The antibacterial activity of Paracin1.7 was regulated by the quorum-sensing system in HD1.7．