Abstract:Abstract:To investigate the functions of the bacteria endogenous plasmid，which include bacterial drug resistance，symbiosis，capsular formation and heavy metal resistance，the endogenous plasmid needs to be cured first． We reviewed physical，chemical and molecular biological methods of endogenous plasmid curing，clarified the curing principles．The prospective of research on plasmid curing was also discussed，based on our own studies．

Abstract:Abstract:With the development of molecular biological techniques and progress of sequencing virus genome，scientists pay great attentions tothe genetic diversity of viruses，which are ubiquitous and abundant in natural environments．So far，no universal genetic marker，analogous to 16S rDNA and 18S rDNAused for microbial communities exists throughout all viruses．However，some family-specific genes encodingconserved amino acids have been proposed for the evaluation of phage diversity and a series of breakthroughachievements were obtained．In this paper，we targeted the capsid assembly protein genes (g20) of cyanophages and reviewed the recent progress on their genetic diversity in natural environments of marines，lakes and paddy fields and discussed the relationship between distribution of g20 gene of cyanophages and its environments．Those studies showed that the distribution of g20 gene varied with environments and many unique clusters were found in different natural environment． In final，several research issues and the future research tendencies for the study of environmental g20 gene were also addressed in this paper．

Abstract:Abstract:［Objective］We assessed the culturable bacterial diversity in tin mine area in Gejiu，Yunnan，China，and heavy-metal tolerance of the isolated bacteria．［Methods］Bacterial strains were isolated from the samples by using the culture-dependent method and investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons．Representatives of isolates were selected to detect their tolerance against heavy metals of Pb2 + and Cd2 +．［Results］We isolated 214 bacterial strains from soils by using two media． Based on colony characteristics，we selected 107 representatives of strains for phylogenetic analysis based on 16S rRNA gene sequences．Results showed that 107 isolates belong to 42 genera of 25 families of 12 orders in 5 phyla．The most abundant isolates were within the phylum Proteobacteria (69.2%) and the genus Pseudomonas (24.8%)．Among the 107 isolates，at least 2 strains should represent 2 potential novel species．One hundred and five strains were selected to investigate heavy-metal tolerance and results showed that 73. 3% and 8. 6% isolates could grow under 1000 mg /L Pb2+ and Cd2+ ，respectively． Most strains were more sensitive to Cd2 + than Pb2 +．Isolates from tin mine tunnel adit were more sensitive to Cd2+ or Pb2+ than those from tin tailings area．Moreover，2 strains (DT47-2A and DT50-1) can grow under both 1000 mg/L Cd2+ and Pb2+．［Conclusion］There is abundant bacteria as well as source of novel taxa in Gejiu tin mining area．Most of them had the ability of high resistance against heavy-metal Cd2+ or Pb2+ ．

Abstract:Abstract: ［Objective］ To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo ofexpression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv．oryzae，the bacterial leaf blight pathogen of rice．［Methods］A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-β-D-galactoside (IPTG) induction． The recombinant protein was purified by Ni-NTA resin． The binding affinity between purified Clpxoo protein and the promoter of endoglucanase gene (engAxoop) was determined by electrophoretic mobility shift assay using fluoresce in (FAM) -labeled probes．The role of c-di-GMP on the binding was also examined．［Results］Under the optimized conditions，Clpxoo was expressed and purified successfully． Mobility shift of engAxoo-p in the presence of Clpxoo was observed，indicating that specific binding occurred between them．Moreover，addition of c-di-GMP molecules in the above reaction system abolished such binding．［Conclusion］ Once interacting with the signal molecule c-di-GMP，Clpxoo conformational structure may change substantially，which results in inhibition of binding to engAxoo-p; The optimized methods for Clpxoo protein purification and electrophoretic mobility shift assay (EMSA) can be used for subsequent identification of Clp regulon in a larger scale．

Abstract:Abstract:［Objective］ To determine the taxonomic position of mineral-weathering bacterium L11 isolated from soil of potassium mine tailing of Nanjing and to elucidate the weathering mechanism of the strain，which will offer the basis for the interaction between microorganism and mineral．［Methods］16S rRNA gene was sequenced and neighbor-joining phylogenetic tree was constructed to identify strain L11．The ability of strain L11 to weather potash feldspar was evaluated by shaking culture.Scanning electron microscope and Energy-dispersive spectrometry were used to observe the mineral weathering and to analyze the elements of mineral surface，respectively．Mineral (＜2μm in diameter) was determined by X-ray diffraction.［Results］Phylogenetic analysis of strain L11 based on 16S rRNA gene sequence was closest to Bacillus altitudinis (99.9%) ．Mineral dissolution experiments showed that strain L11 dissolved potash feldspar and significantly released more Si，Al and Fe elements by producing more organic acids．Many bacteria and some spherical minerals were observed on the surfaces of the feldspar and the energy-dispersive spectrometry analysis showed that the new minerals contained more Fe．After 30 days，siderite might be the newly-formed mineral identified by X-ray diffraction in the mineral weathering process．［Conclusion］ Strain Bacillus sp．L11 could accelerate weathering of potash feldspar， changemineral surface morphologyand induce the formation of new mineral complex.

Abstract:Abstract:［Objective］To explore the role of sgvS in the biosynthesis of 3-hydroxypicolinic acid moiety ofviridogrsein，and to analyze the substrate specificity of 3-hydroxypicolinic acid moiety in the viridogrsein biosynthetic pathway.［Methods］Through gene insertion inactivation and in trans complementation strategies，we obtained the gene inactivation mutant sgvS andits complementation mutant ΔsgvS:: sgvS． Meanwhile， we fed 3-hydroxypicolinic acid， picolinic acid， 2-piperidinecarboxylic acid，3-chloropyridinecarboxylic acid，4-chloropyridinecarboxylic acid，3，5-chloropyridinecarboxylic acid，nicotinic acid，2-chloronicotinic acid，2-fluoronicotinic acid acid，5-fluoronicotinic acid and 6-fluoronicotinic acid to the sgvSmutant，respectively．Thefermentation extracts were analyzed by HPLC.［Results］ sgvS mutant abolished the viridogrsein production; viridogrsein production was restored through in trans complementation of sgvS mutant or by feeding 3-hydroxypicolinic acid to the sgvS mutant．No new viridogrsein analogues were observed by feeding other above mentioned 3-hydroxypicolinic acid analogues tothe ΔsgvS mutant.［Conclusion］sgvS is necessary for the biosynthesis of 3- hydroxypicolinic acid moiety．The biosynthetic protein，SgvD1，activates 3-hydroxypicolinic acid，showing strict substrate specificity en route to the viridogrsein biosynthesis.

Abstract:Abstract:［Objective］We studied the effects of nitrogen concentration on xylose metabolism and organic acid production in Rhizopus oryzae．［Methods］ We studied the effect of different nitrogen concentration in media on biomass，organic acids accumulation，activity of key enzymes ( xylose reductase，XR，glucose-6-phosphate dehydrogenase，G6PD) involved in the metabolism and intracellular redox ratios of R. oryzae.[Results] Both intracellular redox ratios and key enzymes activities stayed low，resulting in 4.02 g/L biomass，6.55 g/L fumaric acid production and high concentration of residual xylose under nitrogen-limitation conditions (0.15 g/L urea). Intracellular redox ratios and activity of key enzymes increased under rich nitrogen culture (2.4 g/L urea)，leading to high biomass 18.01 g/L，high xylose uptake speed 2.03 g/(L·h) but low fumaric acid production (0.27 g/L)．When concentration of urea was 0.6 g/L，biomass 9.11 g/L and fumaric acid 12.28 g/L，NADPH /NADP+，XR and G6PD activities were medium whereas NADH /NAD + reached the highest compared to those in rich nitrogen or nitrogen limitation.［Conclusion］Sufficient nitrogen source strengthened the xylose metabolic activity by promoting the key enzymes activity and intracellular redox ratios in R. oryzae. Optimized nitrogen concentration will enhance fumaric acid production with R. oryzae.

Abstract:Abstract:［Objective］ High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed，which could be used for other applications of S. cerevisiae in metabolic engineering．［Method］ We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5，Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A．The URA3 + transformants were selected．By comparing the difference in the mean florescence value of mCherry in transformants，the effect of three promoters was detected in the co-expression cassette．The copy numbers of the interested genes in the genome were determined by Real-Time PCR．We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure． To verify the application of co-expression cassette，the ORF of mCherry was replaced by β-galactosidase (LACZ) and xylose reductase (XYL1)．The enzyme activities and production of β-galactosidase and xylose reductase were detected.［Result］ mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter． The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5．The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES．The highest enzyme activity was 0.209 U/mg crude protein in the transformants WIX-10.β-galactosidase was also expressed successfully．The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein.［Conclusion］ The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae．This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.

Abstract:Abstract:［Objective］The aims of this study were to investigate yeast diversity in Yangzonghai Lake，and to explore the value of these yeasts in such ecological environment，and at the same time，to analyze biological factors and abiotic factors on the influence of the yeast diversity．［Methods］Water was filtered through cellulose acetate membrane，serial dilutions of soil and sediment; The D1/D2 domain of 26S rRNA gene of all yeast strains have been sequenced and analyzed．The ability of the yeast strains to produce various enzymes was tested by enzyme screening plates．［Results］In total of 201 yeast strains were isolated，these strains were identified as 48 species in 15 genera，including 10 presumed new species or variety; 15.9% showed at least one extracellular enzymatic activity，mainly the lipases and amylases．［Conclusion］The yeast community from Yangzonghai Lake showed high species richness，and anthropogenical influenced is an important factor for the yeast count and distribution，at same time，conductivity and turbidity are also important factors for yeast distribution; Some yeasts produced at least one extracellular enzymatic activity，which suggested that these microorganisms are participating in nature cycle and are metabolically active in the lake．

Abstract:Abstract: ［Objective］ A19-ΔVirB12 deletion mutant of Brucella abortus was constructed by using homologous recombination technology．BALB/c mice were vaccinated intraperitoneally with the mutant to evaluate protective efficacy against Brucella abortus 2308 challenge.［Method］ A SacB gene was amplified by PCR from pIB279 plasmid．The sequences upstream and downstream of the VirB12 gene were amplified by PCR from Brucella abortus A19．These three PCR products were subsequently inserted into pBK-CMV vector，namely pBK-CMV-SacB-VirB12．This construct was transformed into Brucella abortus A19．The A19-Δ VirB12 mutants were obtained by Kanr and 5% sucrose selection．Sixweek-old female BALB/c mice were distributed into three treatment groups，includingA19-Δ VirB12 group，A19 group and PBS control group. BALB/c mice were vaccinated intraperitoneally at a dose of 5.0×104 CFU. At the 45-dayspostimmunization，all of mice were challenged with 2308 strain．Fifteen days after thechallenge，the levels of infection were expressed as means of the log10 CFU/spleen values． The histological changes were assessed among the groups．［Results］Compared with PBS control group，the A19-Δ VirB12 deleted mutant had astatistically significant protection against 2308 challengesimilar to A19 strain． Western blotting showed that A19-Δ VirB12 mutant did not express VirB12 protein．［Conclusion］The A19-Δ VirB12 deleted mutantelicits a strong protective immunity，and may becomea promising vaccine candidate.

Abstract:Abstract:［Objective］ To investigate the pathogenicity of tick-borne encephalitis virus (TBEV) to monocytes and its proliferation characteristics.［Methods］After being infected with TBEV，the cytopathic effect of monocyte cell line THP-1 was observed and viral titers were evaluated．Cell culture supernatants at different time points after infection were collected and the nucleic acids of TBE virus and the infection percentage were tested by using Real-time RT-PCR and fluorescence-activated cell sorting (FACS); the survival rate of THP-1 after TBEV infection was detected by Dimethylthiazol-carboxymethoxyp- henyl-Sulfophenyl-2H-tetrazolium inner salt (MTS).［Results］ Real-time RT-PCR and cytopathic assay both demonstrated that TBE virus could replicate in monocytes. The virus particles could be detected and visualized by FACS． In addition，monocyte viability was significantly decreased 5 days after infection with TEBV.［Conclusion］ TBEV can efficiently replicate and proliferate inTHP-1 cells，indicating that monocytes may play an important role in the process of TBEV spreading to various tissues and organs after infection.

Abstract:Abstract: ［Objective］ We established a genetic transformation system for Penicillium brevicompactum to produce mycophenolic acid.［Methods］We developed protoplast transformation methods mediated by Polyethylene glycol，using phleomycin resistance gene (Sh ble) as a dominant selection marker.［Result］The frequency of transformation was up to 2－3 transformants per μg DNA; analysis of the transformants by PCR showed that the foreign DNA had been integrated into the host genome．The transformants retained stable after generation.［Conclusion］The establishment of the genetic transformation system of Penicillium brevicompactum could serve as the basis for the research of molecular biology and the breeding of gene engineering of the fungus.

Abstract:Abstract:［Objective］ To construct a transformation system in Gliocladium sp．6.102，a Cordyceps-colonizing fungus producing a variety of epipolythiodioxopiperazine (ETP) compounds with drug potentials.［Methods］ Agrobacterium tumefaciens-mediated transformation (ATMT) was used to transform Gliocladium sp. 6. 102. The factors of bacterial cell concentration，co-cultivation time，pH and acetosyringone concentration were optimized．［Results］A total of 50－100 transformants per 106 fungal conidia was obtained via the optimal ATMT method．The genes encoding hygromycin B phosphotase and enhanced green fluorescent protein (EGFP) were transferred into Gliocladium sp. by the optimal ATMT method. The marker genes were successfully expressed and stably maintained in the transgenic fungus.［Conclusion］A transformation system was established for Gliocladium sp. 6. 102 and this system may be useful to identify ETP biosynthetic genes in Gliocladium．

Abstract:Abstract:［Objective］ Bacteria producing proteases were isolated and selected from the environment and the alkaline proteases with superior performance for commercial exploitation were screened.［Methods］ The strain producing extracellular proteases was isolated by a casein plate and was identified by biochemical and morphological tests and by 16S rDNA sequence analysis. To acquire the open reading frame (ORF) of the protease，degenerate primers designing and genome walking method were used. The precursor and mature peptide of the protease were recombinant expressed in BL21 (DE3). After purification of the active protease，the characteristics and the catalytic ability were detected using synthetic peptide succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrates.［Results］Strain L010 isolated was named as Bacillus sp. L010 after identification. The ORF of the protease was 1149-bp long and encoded a protein of 382 amino acids comprised with a 30-residual signal peptide，a 77-residual propeptide，and a 275-residual mature protein，and the encoded protein was one of subtilisins-a member of serine proteases and designated as SprD. The precursor of SprD (pro-SprD) autoprocessed into active SprD mediated by the propeptide when pro-SprD was recombinant expressed in BL21 (DE3)．The enzyme exhibited high catalytic efficiency (Kcat/Km) towards synthesis substrates with optimal activity at 70℃ and pH 9－10．SprD was stable over a range of pH 7.0 to 10.0 and was thermal stable at 25℃ － 60℃.［Conclusion］The high stability of SprD towards alkaline conditions (pH 7 － 10) and under temperature 25℃－60℃ and the high catalytic efficiency suggested that the protease would find research value and potential applications.

Abstract:Abstract:Mangroves are woody plants located in tropical and subtropical intertidal coastal regions． Driven by the discovery of novel natural products from marine environment，mangrove is becoming a hot spot for actinomycetes resources collection and secondary metabolites (natural products) identification as well as their biosynthesis mechanism investigation．Salinaspora A produced by a Salinispora strain isolated from Bahamas mangrove environment，is in the first clinical trial．Till the time of writing this paper，24 genera of 11 families and 8 suborders under the actinomycetale have been reported from mangrove，among which 3 are new genera，and 31 are new species．At the same time，secondary metabolites were identified from the mangrove actinomycetes culture，including alkanoids and quinines，azalomycins，antimycins，bezamides and quinazolines，divergolides，indole derivatives，kandenols，macrocyclic dilactones，and the attractive structures，such as the Streptocarbazoles，the multicyclic indolsesquiterpenes，and xiamycin presented unique structures．Their biosynthetic mechanism has also been investigated． Most of the metabolites were isolated from streptomycetes，with a few from Micromonospora and Saccharopolyspora．