Abstract:Peashrub (Caragana korshinskii Kom) is a kind of excellent shrub used for dune-fixation in Loess Plateau of China. In order to explore relationship between peashrub and soil microorganisms，microbial communities diversity associated with rhizoplane，rhizosphere and bulk soil of peashrub in Loess Plateau of China were characterized based on a culture-independent approach. Three 16S rDNA gene libraries were constructed，respectively，and each different profile was used to define an operational taxonomic unit (OTU). The numbers of microorganisms decreased as root proximity decreased and a few OTUs became dominant. Phylogenetic analyses indicated that Proteobacteria was the predominant group in rhizoplane，which included many α-Proteobacteria，partially consisted of rhizobia，and γ-Proteobacteria beneficial to plant growth. In bulk soil，the most frequent OTUs were closely related to Archaea，while Acidobacteria was the dominant group in rhizosphere of peashrub. The diversity index (H′) was higher in rhizosphere than in rhizoplane and bulk soil，whereas microbial populations in rhizoplane and bulk soil had the greater dominance indices (D). It was shown that there was a significant change in microbial species composition along the root gradient，shifting from complex plant-associated bacterial community in the root habitats to a simple bacterial community in the bulk soil. These results showed that plant roots and soil conditions created a selective environment for microbial populations.

Abstract:Peashrub (Caragana korshinskii Kom) is a kind of excellent shrub used for dune-fixation in Loess Plateau of China. In order to explore relationship between peashrub and soil microorganisms，microbial communities diversity associated with rhizoplane，rhizosphere and bulk soil of peashrub in Loess Plateau of China were characterized based on a culture-independent approach. Three 16S rDNA gene libraries were constructed，respectively，and each different profile was used to define an operational taxonomic unit (OTU). The numbers of microorganisms decreased as root proximity decreased and a few OTUs became dominant. Phylogenetic analyses indicated that Proteobacteria was the predominant group in rhizoplane，which included many α-Proteobacteria，partially consisted of rhizobia，and γ-Proteobacteria beneficial to plant growth. In bulk soil，the most frequent OTUs were closely related to Archaea，while Acidobacteria was the dominant group in rhizosphere of peashrub. The diversity index (H′) was higher in rhizosphere than in rhizoplane and bulk soil，whereas microbial populations in rhizoplane and bulk soil had the greater dominance indices (D). It was shown that there was a significant change in microbial species composition along the root gradient，shifting from complex plant-associated bacterial community in the root habitats to a simple bacterial community in the bulk soil. These results showed that plant roots and soil conditions created a selective environment for microbial populations.

Abstract:Soil and sediment samples were collected from saline and alkaline soils or lakes in the Qinghai Province，northwestern China. 145 actinomycete strains were isolated using Glucose-Peptone-Yeast extract agar (GPY) and ISP medium 2 agar supplemented with 1.0～3.0mol/L NaCl at pH 7.5～10. The antitumor activities in vitro of the fermentation broth extracts from the 145 test strains were detected in 6 human tumor cell lines (gastric cancer GXF251L，lung cancer LXFL529L，mammary cancer MAXF401NL，melanoma cancer MEXF462NL，renal cancer RXF486L and uterus cancer UXF1138L). Out of 145 test strains，26 strains were positive in antitumor activities（17.9%），among them 19 strains belong to the genus Nocardiopsis，7 strains belong to the genus Streptomyces. Then 8 antitumor-positive strains were submitted for 16S rRNA gene amplification and phylogenetic analysis after a comparison of antitumor activities，morphological，physiological characteristics and whole cell amino acids analysis. The results suggested that strain YIM 80139 is a member of a known Streptomyces species S. griseus，while strain YIM 80038 may represent a potential new Streptomyce species，and that the other 6 strains may represent 4 potential new species of the genus Nocardiopsis. The results presented above showed that actinomycetes isolated from saline and alkaline samples are important resources for bioactive compounds，and the abundant microbial diversity in the saline and alkaline environments in the Qinghai Province，Northwestern China is attractive for further investigation.

Abstract:Soil and sediment samples were collected from saline and alkaline soils or lakes in the Qinghai Province，northwestern China. 145 actinomycete strains were isolated using Glucose-Peptone-Yeast extract agar (GPY) and ISP medium 2 agar supplemented with 1.0～3.0mol/L NaCl at pH 7.5～10. The antitumor activities in vitro of the fermentation broth extracts from the 145 test strains were detected in 6 human tumor cell lines (gastric cancer GXF251L，lung cancer LXFL529L，mammary cancer MAXF401NL，melanoma cancer MEXF462NL，renal cancer RXF486L and uterus cancer UXF1138L). Out of 145 test strains，26 strains were positive in antitumor activities（17.9%），among them 19 strains belong to the genus Nocardiopsis，7 strains belong to the genus Streptomyces. Then 8 antitumor-positive strains were submitted for 16S rRNA gene amplification and phylogenetic analysis after a comparison of antitumor activities，morphological，physiological characteristics and whole cell amino acids analysis. The results suggested that strain YIM 80139 is a member of a known Streptomyces species S. griseus，while strain YIM 80038 may represent a potential new Streptomyce species，and that the other 6 strains may represent 4 potential new species of the genus Nocardiopsis. The results presented above showed that actinomycetes isolated from saline and alkaline samples are important resources for bioactive compounds，and the abundant microbial diversity in the saline and alkaline environments in the Qinghai Province，Northwestern China is attractive for further investigation.

Abstract:During the summer of 2006，an epizootic occurred among cultured tongue sole (Cynoglossus semilaevis Gunther) in a fish farm in Qingdao，China. The diseased tongue sole exhibited haemorrhaging of the basal fin，yellowed kidney and ulceration of the body surface. A Gram-negative，rod shaped bacterium (designated strain WY06) was isolated from the gall bladder of diseased fish. Pathogenicity assays revealed that WY06 was virulent to tongue sole and zebrafish (Danio rerio) by intraperitoneal injection challenge，with the LD₅₀ being calculated as 5.5×10³ cfu/g of fish (5.2×10⁵ cfu/fish) and 1.9×10³ cfu/g of fish (8.9×10² cfu/fish) respectively. The 16S rRNA gene sequence of strain WY06 demonstrated high similarity (99%) with Photobacterium damselae subsp. piscicida. Phylogenetic analysis showed a clear association of strain WY06 with P. damselae subsp. piscicida. Additional evidence of the identification included in morphological，physiological and biochemical data. The pathogen was sensitive to cifuroxime (３０μg) and ceftriaxone sodium (３０μg). Present study describes P. damselae subsp. piscicida from diseased fish for the first time in China.

Abstract:During the summer of 2006，an epizootic occurred among cultured tongue sole (Cynoglossus semilaevis Gunther) in a fish farm in Qingdao，China. The diseased tongue sole exhibited haemorrhaging of the basal fin，yellowed kidney and ulceration of the body surface. A Gram-negative，rod shaped bacterium (designated strain WY06) was isolated from the gall bladder of diseased fish. Pathogenicity assays revealed that WY06 was virulent to tongue sole and zebrafish (Danio rerio) by intraperitoneal injection challenge，with the LD₅₀ being calculated as 5.5×10³ cfu/g of fish (5.2×10⁵ cfu/fish) and 1.9×10³ cfu/g of fish (8.9×10² cfu/fish) respectively. The 16S rRNA gene sequence of strain WY06 demonstrated high similarity (99%) with Photobacterium damselae subsp. piscicida. Phylogenetic analysis showed a clear association of strain WY06 with P. damselae subsp. piscicida. Additional evidence of the identification included in morphological，physiological and biochemical data. The pathogen was sensitive to cifuroxime (３０μg) and ceftriaxone sodium (３０μg). Present study describes P. damselae subsp. piscicida from diseased fish for the first time in China.

Abstract:Contagious caprine pleuropneumonia (CCPP) is caused by Mycoplasma capricolum subsp.capripneumoniae (Mccp). The aims of this study were to identify 4 Chinese isolated strains employing molecular methods and to determine the appropriate subspecies classification of these strains. Three genome fragments (A，B and C) from each strain were amplified and then transformed into plasmids. The inserted fragments were sequenced and analyzed by comparison with six members of the Mycoplasma mycoides cluster. Cleavage of the PCR products of the 4 strains with PstI yielded three fragments 548，420 and 128bp in length，just like strain F38. The other M. mycoides cluster members had only 2 fragments of 428 and 128bp. Homology analysis of fragment B indicated that the 4 strains exhibited 99.5% homology with Mccp reference strain F38，98.9% with M. capricolum subsp. Capricolum (Mcc) strain California Kid，and only 95.4% with Mmc strain ZZ. In fragment C，the 4 strains had 67.4%～67.6% homology with Mmc PG3，95.1%～98.6% with Mcc strains 8601-50 and California Kid，99.6%～99.8% with Mccp strains 97097ET，Gabes and F38. The analysis revealed that 4 pathogeny strains，87001，87002，367，1653，isolated from China are more closely related to Mccp than to Mcc. Therefore the pathogeny of CCPP in China should be reclassified as Mccp.

Abstract:Contagious caprine pleuropneumonia (CCPP) is caused by Mycoplasma capricolum subsp.capripneumoniae (Mccp). The aims of this study were to identify 4 Chinese isolated strains employing molecular methods and to determine the appropriate subspecies classification of these strains. Three genome fragments (A，B and C) from each strain were amplified and then transformed into plasmids. The inserted fragments were sequenced and analyzed by comparison with six members of the Mycoplasma mycoides cluster. Cleavage of the PCR products of the 4 strains with PstI yielded three fragments 548，420 and 128bp in length，just like strain F38. The other M. mycoides cluster members had only 2 fragments of 428 and 128bp. Homology analysis of fragment B indicated that the 4 strains exhibited 99.5% homology with Mccp reference strain F38，98.9% with M. capricolum subsp. Capricolum (Mcc) strain California Kid，and only 95.4% with Mmc strain ZZ. In fragment C，the 4 strains had 67.4%～67.6% homology with Mmc PG3，95.1%～98.6% with Mcc strains 8601-50 and California Kid，99.6%～99.8% with Mccp strains 97097ET，Gabes and F38. The analysis revealed that 4 pathogeny strains，87001，87002，367，1653，isolated from China are more closely related to Mccp than to Mcc. Therefore the pathogeny of CCPP in China should be reclassified as Mccp.

Abstract:Based on our established infectious clone of PRRSV，designated as pCBC2，a series of mutagenesis of 3′-untranslated region (3′-UTR) at primary structure and secondary structure level were constructed. Then the full length mutant clones were transfected into MARC-145 cells，from which the influences of the discrete 3′-UTR mutation on PRRSV replication and transcription were analyzed. The properties of the rescued mutant viruses were then further characterized by Northern Blot and plaque morphology analysis. Our results demonstrated that PRRSV could tolerate more than 41 nucleotides deletion and 23nt insertion in the 3′-UTR，however，minor changes in the conserved stem loop region destroyed virus infectivity. To sum up，the stem-loop structure was essential for virus viability，but 5′ end of the 3′-UTR tolerates certain level of nucleotide deletion or insertion. This is the first report to define the essential sequence and secondary structure for PRRSV genome replication and it is useful for future research about the regulation element.

Abstract:Based on our established infectious clone of PRRSV，designated as pCBC2，a series of mutagenesis of 3′-untranslated region (3′-UTR) at primary structure and secondary structure level were constructed. Then the full length mutant clones were transfected into MARC-145 cells，from which the influences of the discrete 3′-UTR mutation on PRRSV replication and transcription were analyzed. The properties of the rescued mutant viruses were then further characterized by Northern Blot and plaque morphology analysis. Our results demonstrated that PRRSV could tolerate more than 41 nucleotides deletion and 23nt insertion in the 3′-UTR，however，minor changes in the conserved stem loop region destroyed virus infectivity. To sum up，the stem-loop structure was essential for virus viability，but 5′ end of the 3′-UTR tolerates certain level of nucleotide deletion or insertion. This is the first report to define the essential sequence and secondary structure for PRRSV genome replication and it is useful for future research about the regulation element.

Abstract:Type Ⅲ secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins，chaperons，regulators，and effectors proteins，respectively. Although some genes have been studied previously，functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First，by characterization of the phenotypes of pcr2- mutant，we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein，suggesting that Pcr2 protein involved in both the secretion and translocation processes of TTSS. Second，evidences were provided that no PopN protein was detectable in supernatant of pcr2- mutant culture. Combined with the data from the bacterial two-hybrid system，we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third，Pcr2 protein was found secreted in a TTSS-dependent manner，suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.

Abstract:Type Ⅲ secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins，chaperons，regulators，and effectors proteins，respectively. Although some genes have been studied previously，functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First，by characterization of the phenotypes of pcr2- mutant，we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein，suggesting that Pcr2 protein involved in both the secretion and translocation processes of TTSS. Second，evidences were provided that no PopN protein was detectable in supernatant of pcr2- mutant culture. Combined with the data from the bacterial two-hybrid system，we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third，Pcr2 protein was found secreted in a TTSS-dependent manner，suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.

Abstract:In Saccharomyces cerevisiae，protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man₈GlcNAc₂-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man_8-13GlcNAc₂ structure. Alternatively，core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of α1，6-mannosyl residues with branched α1，2- and α1，3-mannosyl side chains. Mnn1p and Och1p play an important role in this process. The null disruption of MNN1，OCH1 was replaced by the S. cerevisiae URA3，HIS3，respectively. To characterize the N-glycosylation in the mnn1 och1 mutant，mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol，and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion，and then peptides and detergents were removed by passage through ion exchange columns. For desalting，glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH2 column，and result showed dominantly a single peak. MALDI TOF/MS analysis ofthis peak revealed that its molecular weight was 1796.5Da，which corresponds to the calculated mass of Man₈GlcNAc₂-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans，and glycoproteins were glycosylated with a single core type glycan，Man₈GlcNAc₂，in the mnn1 och1 mutant.

Abstract:In Saccharomyces cerevisiae，protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man₈GlcNAc₂-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man_8-13GlcNAc₂ structure. Alternatively，core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of α1，6-mannosyl residues with branched α1，2- and α1，3-mannosyl side chains. Mnn1p and Och1p play an important role in this process. The null disruption of MNN1，OCH1 was replaced by the S. cerevisiae URA3，HIS3，respectively. To characterize the N-glycosylation in the mnn1 och1 mutant，mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol，and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion，and then peptides and detergents were removed by passage through ion exchange columns. For desalting，glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH2 column，and result showed dominantly a single peak. MALDI TOF/MS analysis ofthis peak revealed that its molecular weight was 1796.5Da，which corresponds to the calculated mass of Man₈GlcNAc₂-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans，and glycoproteins were glycosylated with a single core type glycan，Man₈GlcNAc₂，in the mnn1 och1 mutant.

Abstract:The fed operon gene clusters with each size of 5.6kb，encoding the F18ab or F18ac fimbriae，was amplified respectively by high fidelity PCR using the genomic DNA templates from F18 fimbriae E. coli strains 107/86 or 2134P. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pET-22b(+)，the recombinant plamids with the inserts of both type of fed gene clusters were constructed and screened，further confirmed by the means of combination with restriction endonuclease analysis and sequencing. The both types of fimbriae F18ab and F18ac were expressed efficiently in the E. coli BL21(DE3) after proper concentration of IPTG induction. Expressed fimbriae were revealed and comfirmed by transmissible electromicroscope observation. The both fimbriae F18ab and F18ac were isolated and purified from the recombinant E. coli，and only a single major band of protein with size of approximately 15kDa was visualized in Coomassie blue-stained gels after SDS-PAGE. The rabbits sera with high titer of anti-F18 fimbriae were detected after being immunized with the purified F18ab or F18ac fimbriae. The results of combination of agglutination assay with Western blotting showed that the sera directed against both fimbriae F18ab and F18ac reacted positively with the F18 fimbriae from both wild E. coli 107/86 and 2134P.Small intestine epithelial cells with F18 fimbriae receptors，which were from post-weaning piglets with the genotypes of FUT1 gene both M307GG and M307AG，were prepared and tested for the adherence of E. coli expressing F18 fimbriae under the microscopic examination. Adhesion and adhesion inhibition test showed both of the recombinant E. coli expressing F18ab or F18ac fimbriae respectively could adhere to the jejunal epithelial cells in vitro as E. coli 107/86 and 2134p did. The both of anti-sera directed against fimbriae F18ab or F18ac respectively can efficiently inhibit the fimbriae-mediated post-weaning piglet jejunal epithelial cells adherence to both the recombinant E. coli (expressing F18ab or F18ac fimbriae) and wild type E. coli (107/86 and 2134P).

Abstract:The fed operon gene clusters with each size of 5.6kb，encoding the F18ab or F18ac fimbriae，was amplified respectively by high fidelity PCR using the genomic DNA templates from F18 fimbriae E. coli strains 107/86 or 2134P. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pET-22b(+)，the recombinant plamids with the inserts of both type of fed gene clusters were constructed and screened，further confirmed by the means of combination with restriction endonuclease analysis and sequencing. The both types of fimbriae F18ab and F18ac were expressed efficiently in the E. coli BL21(DE3) after proper concentration of IPTG induction. Expressed fimbriae were revealed and comfirmed by transmissible electromicroscope observation. The both fimbriae F18ab and F18ac were isolated and purified from the recombinant E. coli，and only a single major band of protein with size of approximately 15kDa was visualized in Coomassie blue-stained gels after SDS-PAGE. The rabbits sera with high titer of anti-F18 fimbriae were detected after being immunized with the purified F18ab or F18ac fimbriae. The results of combination of agglutination assay with Western blotting showed that the sera directed against both fimbriae F18ab and F18ac reacted positively with the F18 fimbriae from both wild E. coli 107/86 and 2134P.Small intestine epithelial cells with F18 fimbriae receptors，which were from post-weaning piglets with the genotypes of FUT1 gene both M307GG and M307AG，were prepared and tested for the adherence of E. coli expressing F18 fimbriae under the microscopic examination. Adhesion and adhesion inhibition test showed both of the recombinant E. coli expressing F18ab or F18ac fimbriae respectively could adhere to the jejunal epithelial cells in vitro as E. coli 107/86 and 2134p did. The both of anti-sera directed against fimbriae F18ab or F18ac respectively can efficiently inhibit the fimbriae-mediated post-weaning piglet jejunal epithelial cells adherence to both the recombinant E. coli (expressing F18ab or F18ac fimbriae) and wild type E. coli (107/86 and 2134P).

Abstract:To further investigate into the prevalence of F1 fimbriae and high pathogenicity island (HPI) in avian Escherichia coli，a total of 69 bacteria isolates (29 from geese and 40 from chickens) were obtained from deceased poultry and characterized to be Escherichia coli by gram staining，culture characterizing and bio-chemical testing. Two sets of primers were designed or analyzed with DNAStar software based on the F1 and HPI sequences that deposited in GenBank and synthesized by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai，P. R. China). All the primers were tested for their specificities by using reference E. coli strains，and it is confirmed that the PCR using primers F1-F and F1-R could identify F1⁺ E. coli，as well as the PCR using primers irp-F and irp-R could identify HPI⁺ E. coli. All the isolates were submitted to PCR detection，and the data shows 46 isolates (66.7%) were F1-positive，10 isolates (14.5%) were F1⁺HPI-positive and 2 isolates (20.0%) were HPI-positive. Furthermore analysis indicated that the prevalence of F1 and HPI have no different between the isolates from geese and chickens，and no different among the isolates from different tissue，such as liver，lung and duodenum. In addition，all the 69 E. coli isolates serological typed，and the results show that the isolates from geese were belonged to O26(25.0%)，O78(12.5%)，O18(12.5%) and O117(12.5%%)，while E coli from chickens were fell into O109(37.5%)，O24(18.75%)，O18(12.5%)，O139(12.5%) and O78(6.25%). Seven kinds of antibiotics were tested on 11 isolates，and it revealed that most isolates were sensitive to cefazolin，nitrofurantoin and gentamycin，while resistant to lincomycin，tetracycline and polymycin B.

Abstract:To further investigate into the prevalence of F1 fimbriae and high pathogenicity island (HPI) in avian Escherichia coli，a total of 69 bacteria isolates (29 from geese and 40 from chickens) were obtained from deceased poultry and characterized to be Escherichia coli by gram staining，culture characterizing and bio-chemical testing. Two sets of primers were designed or analyzed with DNAStar software based on the F1 and HPI sequences that deposited in GenBank and synthesized by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai，P. R. China). All the primers were tested for their specificities by using reference E. coli strains，and it is confirmed that the PCR using primers F1-F and F1-R could identify F1⁺ E. coli，as well as the PCR using primers irp-F and irp-R could identify HPI⁺ E. coli. All the isolates were submitted to PCR detection，and the data shows 46 isolates (66.7%) were F1-positive，10 isolates (14.5%) were F1⁺HPI-positive and 2 isolates (20.0%) were HPI-positive. Furthermore analysis indicated that the prevalence of F1 and HPI have no different between the isolates from geese and chickens，and no different among the isolates from different tissue，such as liver，lung and duodenum. In addition，all the 69 E. coli isolates serological typed，and the results show that the isolates from geese were belonged to O26(25.0%)，O78(12.5%)，O18(12.5%) and O117(12.5%%)，while E coli from chickens were fell into O109(37.5%)，O24(18.75%)，O18(12.5%)，O139(12.5%) and O78(6.25%). Seven kinds of antibiotics were tested on 11 isolates，and it revealed that most isolates were sensitive to cefazolin，nitrofurantoin and gentamycin，while resistant to lincomycin，tetracycline and polymycin B.

Abstract:In order to understand the replication kinetics of classical swine fever virus(CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37℃ in 5% CO₂ environment when growing to 80% confluence，the cells were infected with CSFV strain Shimen at 100 TCID₅₀ per well. At various time post infection(p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay(IFA)，RNA replication using reverse transcription real-time PCR and viral production using titration of TCID₅₀. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining.the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However，the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID₅₀ demonstrated that the production of live virions increased at 8h and peaked between 48-72 hrs p.i. without significant lose of titer.

Abstract:In order to understand the replication kinetics of classical swine fever virus(CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37℃ in 5% CO₂ environment when growing to 80% confluence，the cells were infected with CSFV strain Shimen at 100 TCID₅₀ per well. At various time post infection(p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay(IFA)，RNA replication using reverse transcription real-time PCR and viral production using titration of TCID₅₀. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining.the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However，the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID₅₀ demonstrated that the production of live virions increased at 8h and peaked between 48-72 hrs p.i. without significant lose of titer.

Abstract:An H3N2 subtype swine influenza virus，A/Swine/ Guangdong/01/2005(H3N2)，was isolated from pigs with influenza-like signs in Guangdong province in 2005. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the gene segments for sequencing analysis. Phylogenetic analysis showed that the hemagglutinin(HA) gene of A/Swine/ Guangdong/01/2005 shared high degree of sequence identity with those of H3N2 viruses isolated from swine in Guangdong province from 2003 to 2004 and H3N2 viruses isolated from human in New York in the end of 1990s. The neuraminidase (NA) gene of this H3N2 swine virus had high degree of sequence identity with those of H3N2 viruses isolated from human in New York from 1998 to 2000. While the nonstructure (NS) gene and matrix (M) gene of this H3N2 swine virus showed high degree of sequence identity with those of classical H1N1 swine influenza viruses. These results indicated that the M and NS gene might originate from the H1N1 subtype swine influenza viruses，while the HA，NA and other genes of this H3N2 subtype swine influenza virus might originate from the H3N2 subtype human influenza viruses. Therefore，the H3N2 swine influenza virus，A/Swine/Guangdong/ 01/2005(H3N2)，is a recombinant of H3N2 human influenza virus and classical H1N1swine influenza virus.

Abstract:An H3N2 subtype swine influenza virus，A/Swine/ Guangdong/01/2005(H3N2)，was isolated from pigs with influenza-like signs in Guangdong province in 2005. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the gene segments for sequencing analysis. Phylogenetic analysis showed that the hemagglutinin(HA) gene of A/Swine/ Guangdong/01/2005 shared high degree of sequence identity with those of H3N2 viruses isolated from swine in Guangdong province from 2003 to 2004 and H3N2 viruses isolated from human in New York in the end of 1990s. The neuraminidase (NA) gene of this H3N2 swine virus had high degree of sequence identity with those of H3N2 viruses isolated from human in New York from 1998 to 2000. While the nonstructure (NS) gene and matrix (M) gene of this H3N2 swine virus showed high degree of sequence identity with those of classical H1N1 swine influenza viruses. These results indicated that the M and NS gene might originate from the H1N1 subtype swine influenza viruses，while the HA，NA and other genes of this H3N2 subtype swine influenza virus might originate from the H3N2 subtype human influenza viruses. Therefore，the H3N2 swine influenza virus，A/Swine/Guangdong/ 01/2005(H3N2)，is a recombinant of H3N2 human influenza virus and classical H1N1swine influenza virus.

Abstract:The changes of genes expression in HeLa cell during the invasion with Shigella species for 1h and 3h were analyzed by cDNA microarrays. The data showed that the expression levels of 752 genes were altered twice or greater as compared with the control 509 of them were up-regulated，and 306 were down-regulated. It was supposed that some signal pathways in HeLa cell were activated，then many genes were induced，and at last comprehensive cell responses were produced，so that HeLa cell could prevent against Shigella species infection. Two striking difference cDNA fragments TNFR 1B and ERBB2，which were up-regulated in the host epithelial cell during Shigella infection，analyzed expression by real time RT-PCR as determined by cDNA arrays. We suggested they play important roles in response to the invasive S. flexneri 2457T. Based on the results of gene expression alterations，the molecular pathogenic mechanism of Shigella species could be greatly and deeply understood，and the strategy for prevention against and treatment for shigellosis would be formed.

Abstract:The changes of genes expression in HeLa cell during the invasion with Shigella species for 1h and 3h were analyzed by cDNA microarrays. The data showed that the expression levels of 752 genes were altered twice or greater as compared with the control 509 of them were up-regulated，and 306 were down-regulated. It was supposed that some signal pathways in HeLa cell were activated，then many genes were induced，and at last comprehensive cell responses were produced，so that HeLa cell could prevent against Shigella species infection. Two striking difference cDNA fragments TNFR 1B and ERBB2，which were up-regulated in the host epithelial cell during Shigella infection，analyzed expression by real time RT-PCR as determined by cDNA arrays. We suggested they play important roles in response to the invasive S. flexneri 2457T. Based on the results of gene expression alterations，the molecular pathogenic mechanism of Shigella species could be greatly and deeply understood，and the strategy for prevention against and treatment for shigellosis would be formed.

Abstract:Tilletia indica Mitra，an important pest around the world which is the causative agent of Karnal Bunt of wheat，was very close to Tilletia walkeri phylogenetically and morphologically. 69.6ng mitochondrial DNA (mtDNA) of Tilletia indica obtained from 6.4mg total DNA by the method of CsCl/bisbenzimide density gradient ultracentrifugation can be used for electrophoresis，cloning，enzyme restriction analysis and PCR amplification. The ATP6 gene sequence was cloned from the fragment of mtDNA and sequenced for analysis of phylogenetic tree with the other related sequences in GenBank by the software PAUP to reveal the phylogenetic relationships within the Tilletia species.

Abstract:Tilletia indica Mitra，an important pest around the world which is the causative agent of Karnal Bunt of wheat，was very close to Tilletia walkeri phylogenetically and morphologically. 69.6ng mitochondrial DNA (mtDNA) of Tilletia indica obtained from 6.4mg total DNA by the method of CsCl/bisbenzimide density gradient ultracentrifugation can be used for electrophoresis，cloning，enzyme restriction analysis and PCR amplification. The ATP6 gene sequence was cloned from the fragment of mtDNA and sequenced for analysis of phylogenetic tree with the other related sequences in GenBank by the software PAUP to reveal the phylogenetic relationships within the Tilletia species.

Abstract:It is believed that genetic recombination of the endophytes with the hosts that occurred in evolutionary time could result in some endophytes producing certain phytochemical originally characteristic of the host. Based on this widely accepted hypothesis, there have been increasing research efforts focused on screening for novel natural products from endophytes. In this study, antimicrobial and antitumor activities of 165 actinomycetes isolated from medicinal plants collected from Xishuangbanna were tested by agar diffusion method and WST-8 assay respectively. The results showed that over 42% of the isolates exhibited antagonism against pathogenic strains， and 54.5% displayed excellent inhibition against mouse melanoma cell line B16 or/and human alveolar epithelial cell line A549. These results are superior to those of soil actinomycetes, indicating tremendous potential of endophytic of actinomycetes for exploration. Six compounds that had both antimicrobial and antitumor activities were separated and purified from isolate Streptomyces sp. D62 by resin adsorption, silica-gel column and sephadex chromatography, etc. On the basis of spectral analyses, they were identified as antimycin A_4a(1), antimycin A_7a(2), antimycin A_2a(3), antimycin A_1a(4), 10-hydroxy-10-methyl-dodec-2-en-1,4-olide(5) and 6-(2-(4-aminophenyl)-2-oxoethyl)-3,5-dimethyl-tetrahydropyran-2-one(6), with the last one defined as a novel compound. Based on all these results, it is convinced that endophytic actinomycetes are a promising resource for bioactive natural product discovery.

Abstract:It is believed that genetic recombination of the endophytes with the hosts that occurred in evolutionary time could result in some endophytes producing certain phytochemical originally characteristic of the host. Based on this widely accepted hypothesis, there have been increasing research efforts focused on screening for novel natural products from endophytes. In this study, antimicrobial and antitumor activities of 165 actinomycetes isolated from medicinal plants collected from Xishuangbanna were tested by agar diffusion method and WST-8 assay respectively. The results showed that over 42% of the isolates exhibited antagonism against pathogenic strains， and 54.5% displayed excellent inhibition against mouse melanoma cell line B16 or/and human alveolar epithelial cell line A549. These results are superior to those of soil actinomycetes, indicating tremendous potential of endophytic of actinomycetes for exploration. Six compounds that had both antimicrobial and antitumor activities were separated and purified from isolate Streptomyces sp. D62 by resin adsorption, silica-gel column and sephadex chromatography, etc. On the basis of spectral analyses, they were identified as antimycin A_4a(1), antimycin A_7a(2), antimycin A_2a(3), antimycin A_1a(4), 10-hydroxy-10-methyl-dodec-2-en-1,4-olide(5) and 6-(2-(4-aminophenyl)-2-oxoethyl)-3,5-dimethyl-tetrahydropyran-2-one(6), with the last one defined as a novel compound. Based on all these results, it is convinced that endophytic actinomycetes are a promising resource for bioactive natural product discovery.

Abstract:Micrococcu luteus CN1 was found to be able to utilize cyclohexanone well from the strains originally isolated from Pacific Ocean sediment. The optimum conditions for its growth were determined as 25℃-37℃，pH 8，salinity 6%. It could survive in the medium with high concentration of cyclohexanone (>44% V/V), and grew most vigorously in medium with 16.7%(V/V) cyclohexanone. CN1 could transform cyclohexanol to cyclohexanone which could be further degraded and mineralized quickly. This indicated the presence of cyclohexanol dehydrogenase and probable presence of cyclohexanone monooxygenase. With degenerate PCR for cloning part of cyclohexanone monooxygenase gene,the DNA fragment of 450bp was gotten. Amino acid sequence analysis showed that it owned the conserved sequence of the Baeyer-Villiger monooxygenase family and had the highest homology of 80% with cyclohexanone monooxygenase from Arthrobacter sp.BP2, only 53% with that from Acinetobacter sp. NCIMB 9871 which had been the most deeply investigated. So far as we know, both cyclohexanol and cyclohexanone degradation resorted to cyclohexanone monooxygenase. So this gene should be responsible for cyclohexanone degradation in CN1. All the cyclohexanone-degraders previously reported could degrade cyclopentanone, but, CN1 did not degrade cyclopentanone. This indicated that cyclohexanone monooxygenase in CN1 was special. Additionally, it was found for the first time that cyclohexanol could inhibit cyclohexanone degradation to certain degree in CN1.

Abstract:Micrococcu luteus CN1 was found to be able to utilize cyclohexanone well from the strains originally isolated from Pacific Ocean sediment. The optimum conditions for its growth were determined as 25℃-37℃，pH 8，salinity 6%. It could survive in the medium with high concentration of cyclohexanone (>44% V/V), and grew most vigorously in medium with 16.7%(V/V) cyclohexanone. CN1 could transform cyclohexanol to cyclohexanone which could be further degraded and mineralized quickly. This indicated the presence of cyclohexanol dehydrogenase and probable presence of cyclohexanone monooxygenase. With degenerate PCR for cloning part of cyclohexanone monooxygenase gene,the DNA fragment of 450bp was gotten. Amino acid sequence analysis showed that it owned the conserved sequence of the Baeyer-Villiger monooxygenase family and had the highest homology of 80% with cyclohexanone monooxygenase from Arthrobacter sp.BP2, only 53% with that from Acinetobacter sp. NCIMB 9871 which had been the most deeply investigated. So far as we know, both cyclohexanol and cyclohexanone degradation resorted to cyclohexanone monooxygenase. So this gene should be responsible for cyclohexanone degradation in CN1. All the cyclohexanone-degraders previously reported could degrade cyclopentanone, but, CN1 did not degrade cyclopentanone. This indicated that cyclohexanone monooxygenase in CN1 was special. Additionally, it was found for the first time that cyclohexanol could inhibit cyclohexanone degradation to certain degree in CN1.

Abstract:The microbial cooperated reaction is one of the most important forms of microbial degradation of organic pollutants. Although there were many research reports of cooperating degradation, less report on the microbial cooperated of pyrethroid degradation to be found. We have isolated one degrading-bacteria strain named CDT3 for degradation of cypermethrin, which can degraded the cypermethrin into 3-PBA and DCVA. At the same time, we also isolated another degrading-bacteria strain named as PBM11, which could get multiplication on 3-PBA as its C source and energy source. The cooperative degradation process of cypermethrin and 3-Phenoxybenzoic acid (3-PBA) using the two degrading-bacteria strain CDT3 and PBM11 was investigated. An obvious inhibition to the cypermethrin degrading-bacterium strain CDT3(Rhodococcus sp.) by its metabolic mediate 3-PBA was found; meanwhile there is no effect on the growth of 3-PBA degrading-bacterium strain PBM11(Pesudomonas sp.) when the concentration of cypermethrin was lower than 200mg/L. The degradation rate of cypermethrin by both strain CDT3 and PBM11 was higher than that by CDT3 alone. The biomass of PBM11 increased along with the degradation of cypermethrin and 3-PBA, but that of CDT3 not. There was no the accumulation of 3-PBA when the simultaneous addition of strain CDT3 and PBM11, however, an obvious one within 24h if inoculation of strain PBM11 was later 24h after inoculation of strain CDT3, Subsequently the 3-PBA was degraded rapidly by strain PBM11. The strains CDT3 and PBM11 showed some characteristics of co-metabolism, however it is not actual degradation form of co-metabolism. For examples, although the degrading sub product of cypermethrin by CDT3 could be utilized, the multiplication of PBM11 could not enhance the multiplication of CDT3, implied there is no obvious relationship between the two strains. Also, to add PBM11 could eliminate the inhibition of 3-PBA to CDT3. Thus, the cooperating degradation of strains CDT3 and PBM11 could promote the degradation of cypermethrin as well as eliminate the residues of middle products. The present research results provide new evidence for the theory of cooperated degradation, but a further research may be necessary.

Abstract:The microbial cooperated reaction is one of the most important forms of microbial degradation of organic pollutants. Although there were many research reports of cooperating degradation, less report on the microbial cooperated of pyrethroid degradation to be found. We have isolated one degrading-bacteria strain named CDT3 for degradation of cypermethrin, which can degraded the cypermethrin into 3-PBA and DCVA. At the same time, we also isolated another degrading-bacteria strain named as PBM11, which could get multiplication on 3-PBA as its C source and energy source. The cooperative degradation process of cypermethrin and 3-Phenoxybenzoic acid (3-PBA) using the two degrading-bacteria strain CDT3 and PBM11 was investigated. An obvious inhibition to the cypermethrin degrading-bacterium strain CDT3(Rhodococcus sp.) by its metabolic mediate 3-PBA was found; meanwhile there is no effect on the growth of 3-PBA degrading-bacterium strain PBM11(Pesudomonas sp.) when the concentration of cypermethrin was lower than 200mg/L. The degradation rate of cypermethrin by both strain CDT3 and PBM11 was higher than that by CDT3 alone. The biomass of PBM11 increased along with the degradation of cypermethrin and 3-PBA, but that of CDT3 not. There was no the accumulation of 3-PBA when the simultaneous addition of strain CDT3 and PBM11, however, an obvious one within 24h if inoculation of strain PBM11 was later 24h after inoculation of strain CDT3, Subsequently the 3-PBA was degraded rapidly by strain PBM11. The strains CDT3 and PBM11 showed some characteristics of co-metabolism, however it is not actual degradation form of co-metabolism. For examples, although the degrading sub product of cypermethrin by CDT3 could be utilized, the multiplication of PBM11 could not enhance the multiplication of CDT3, implied there is no obvious relationship between the two strains. Also, to add PBM11 could eliminate the inhibition of 3-PBA to CDT3. Thus, the cooperating degradation of strains CDT3 and PBM11 could promote the degradation of cypermethrin as well as eliminate the residues of middle products. The present research results provide new evidence for the theory of cooperated degradation, but a further research may be necessary.

Abstract:The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5α, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.

Abstract:The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5α, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.

Abstract:Two DNA fragments encoding chitinase A and B were amplified from total genomic DNA of Bacillus thuringiensis subsp. colmeri 15A3, and then ligated with pUCm-T cloning vector. The recombinant plasmids pUCm-chiA and pUCm-chiB were transformed into Escherichia coli XL-Blue respectively. Both chiA and chiB were successfully expressed in E. coli with their natural promoters independent of any chitin. Additionally, the expressed ChiA and ChiB could be secreted from E. coli cells. It was proved that two chitinases were constitutively expressed in strain 15A3. The nucleotide sequence of chiA (GenBank Accession Number: EF103273) with a length of 1426bp included an open reading fram(ORF) of 1083 bp encoding for a protein of 360 amino acids . The deduced amino acid sequence showed that the mature protein ChiA with a predicted molecular mass of 36kDa consisted of a single catalytic domain. The nucleotide sequence of chiB (GenBank Accession Number:EF103273) with a length of 2279 bp included an ORF of 2031bp encoding for a protein of 676 amino acid residues . The deduced amino acid sequence showed that the mature protein ChiB with a predicted molecular mass of 70.6kD was composed of three domains： catalytic domain，chitin-binding domain and fibronectin type III-like domain. The comparison of their upstream sequences informed that there were differences between putative promoters of chiA and chiB.

Abstract:Two DNA fragments encoding chitinase A and B were amplified from total genomic DNA of Bacillus thuringiensis subsp. colmeri 15A3, and then ligated with pUCm-T cloning vector. The recombinant plasmids pUCm-chiA and pUCm-chiB were transformed into Escherichia coli XL-Blue respectively. Both chiA and chiB were successfully expressed in E. coli with their natural promoters independent of any chitin. Additionally, the expressed ChiA and ChiB could be secreted from E. coli cells. It was proved that two chitinases were constitutively expressed in strain 15A3. The nucleotide sequence of chiA (GenBank Accession Number: EF103273) with a length of 1426bp included an open reading fram(ORF) of 1083 bp encoding for a protein of 360 amino acids . The deduced amino acid sequence showed that the mature protein ChiA with a predicted molecular mass of 36kDa consisted of a single catalytic domain. The nucleotide sequence of chiB (GenBank Accession Number:EF103273) with a length of 2279 bp included an ORF of 2031bp encoding for a protein of 676 amino acid residues . The deduced amino acid sequence showed that the mature protein ChiB with a predicted molecular mass of 70.6kD was composed of three domains： catalytic domain，chitin-binding domain and fibronectin type III-like domain. The comparison of their upstream sequences informed that there were differences between putative promoters of chiA and chiB.

Abstract:Tetratricopeptide repeat (TPR) domains usually mediate protein-protein interactions. NsdA, one of the 70 proteins containing TPR-like domains in Streptomyces coelicolor A3(2), was previously found to negatively control sporulation and antibiotic production. Here we show that elimination of SCO7252, which encodes another of these proteins, also caused overproduction of two antibiotics, actinorhodin and CDA, but did not affect morphological differentiation. Disruption of SCO1593, encoding another of the family, had no obvious phenotypic effects. In surface-grown cultures, expression of SCO7252, which was named nsdB, was initiated at about 30 h, like that of nsdA. Analysis in silico of the 70 predicted TPR-like-containing proteins of S. coelicolor showed that 32 of them contained only TPR-like domains, and 25 of the remainder contained additional DNA-binding domains, implying that they might control gene expression directly.

Abstract:Tetratricopeptide repeat (TPR) domains usually mediate protein-protein interactions. NsdA, one of the 70 proteins containing TPR-like domains in Streptomyces coelicolor A3(2), was previously found to negatively control sporulation and antibiotic production. Here we show that elimination of SCO7252, which encodes another of these proteins, also caused overproduction of two antibiotics, actinorhodin and CDA, but did not affect morphological differentiation. Disruption of SCO1593, encoding another of the family, had no obvious phenotypic effects. In surface-grown cultures, expression of SCO7252, which was named nsdB, was initiated at about 30 h, like that of nsdA. Analysis in silico of the 70 predicted TPR-like-containing proteins of S. coelicolor showed that 32 of them contained only TPR-like domains, and 25 of the remainder contained additional DNA-binding domains, implying that they might control gene expression directly.

Abstract:The DNA adenine methylase (dam) gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V.parahaemolyticus (96% in identity). The V.harveyi dam gene was subcloned into plasmid pBR322 and the resulting plasmid pBD was introduced into the E.coli strain ER2925 in which the dam gene had been knocked out. DpnⅠ, DpnⅡ, and Sau3AⅠ restriction enzyme analysis of the genomic DNA of ER2925 transformed with pBD indicated that the cloned V.harveyi dam gene could functionally complement the E. coli dam mutant and methylate E.coli chromosome at the GATC sites. The 3251 bp upstream region of V.harveyi dam was obtained by genome walking and analyzed at the sequence level. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length and the other was of 1503 bp in length. The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V. parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V. parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with the shikimate kinase of V. parahaemolyticus. These three genes, together with dam, were arranged in the order of shikimate kinase-3-dehydroquinate synthase-damX-dam. The region immediate upstream of the V.harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named P78, P112，and P477, respectively) and tested for promoter activity. The results showed that，while all the three fragments had detectable promoter activities, the activity of P78 appeared to be higher than that of P₁₁₂ and P₄₇₇.

Abstract:The DNA adenine methylase (dam) gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V.parahaemolyticus (96% in identity). The V.harveyi dam gene was subcloned into plasmid pBR322 and the resulting plasmid pBD was introduced into the E.coli strain ER2925 in which the dam gene had been knocked out. DpnⅠ, DpnⅡ, and Sau3AⅠ restriction enzyme analysis of the genomic DNA of ER2925 transformed with pBD indicated that the cloned V.harveyi dam gene could functionally complement the E. coli dam mutant and methylate E.coli chromosome at the GATC sites. The 3251 bp upstream region of V.harveyi dam was obtained by genome walking and analyzed at the sequence level. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length and the other was of 1503 bp in length. The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V. parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V. parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with the shikimate kinase of V. parahaemolyticus. These three genes, together with dam, were arranged in the order of shikimate kinase-3-dehydroquinate synthase-damX-dam. The region immediate upstream of the V.harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named P78, P112，and P477, respectively) and tested for promoter activity. The results showed that，while all the three fragments had detectable promoter activities, the activity of P78 appeared to be higher than that of P₁₁₂ and P₄₇₇.

Abstract:PCR analysis demonstrated the presence of the ad gene in all 29 S.suis strains tested, but none of the seven S.equi subsp. zooepidemicus strains. The fragment of ad gene of virulent isolate SS2-HA9801 was later cloned into pBAD/Myc-HisC vector via restriction endonuclease and then transformed into host strain TOP10. A recombinant protein of 47000Da was highly expressed after induced by L-ararose and purified by Ni-nitrilotriacetic acid affinity chromatography. Western blotting demonstrated that the recombinant protein can reacted to the polyclonal antibody raised against whole-cell protein of SS2-HA9801, which suggested that it possessed some immunogenicity and may be important for further research. Enzymatic assay revealed that the optimum temperature for its activity is 37℃ and pH is 6.5. Studies with class-specific inhibitors supported the assignment of a sulfhydryl enzyme with some metallo class characteristics.

Abstract:PCR analysis demonstrated the presence of the ad gene in all 29 S.suis strains tested, but none of the seven S.equi subsp. zooepidemicus strains. The fragment of ad gene of virulent isolate SS2-HA9801 was later cloned into pBAD/Myc-HisC vector via restriction endonuclease and then transformed into host strain TOP10. A recombinant protein of 47000Da was highly expressed after induced by L-ararose and purified by Ni-nitrilotriacetic acid affinity chromatography. Western blotting demonstrated that the recombinant protein can reacted to the polyclonal antibody raised against whole-cell protein of SS2-HA9801, which suggested that it possessed some immunogenicity and may be important for further research. Enzymatic assay revealed that the optimum temperature for its activity is 37℃ and pH is 6.5. Studies with class-specific inhibitors supported the assignment of a sulfhydryl enzyme with some metallo class characteristics.

Abstract:On the basis of the sequencing of the N-terminal amino acid of the crystal protein, a nucleotide acid fragment harboring a novel nematicidal gene cry6Aa2 was obtained from Bacillus thuringiensis strain YBT-1518. This fragment contains two ORFs: orf1 and orf2, while a “stem-loop" exists between orf1 and orf2. BLAST showed the similarity of orf1 nucleotide acid sequence with cry6Aa1 is 98%, and has been deposited in the Genbank database (Acc. No. AF499736). The cloning fragment was transferred to crystal negative mutation strain BMB171 by E. coli-Bt shuttle vector pHT304. A 54kDa protein with similarity to strain YBT-1518 was detected in recombinant strain, and rice shaped crystal was detected with transmission electron microscope. Bioassay indicated the LC₅₀ of recombinant strain against second lavae juvenile of Meloidgyne hapla is 9.47μg/mL, nearly equal to the original strain YBT-1518 (10.74μg/mL).

Abstract:On the basis of the sequencing of the N-terminal amino acid of the crystal protein, a nucleotide acid fragment harboring a novel nematicidal gene cry6Aa2 was obtained from Bacillus thuringiensis strain YBT-1518. This fragment contains two ORFs: orf1 and orf2, while a “stem-loop" exists between orf1 and orf2. BLAST showed the similarity of orf1 nucleotide acid sequence with cry6Aa1 is 98%, and has been deposited in the Genbank database (Acc. No. AF499736). The cloning fragment was transferred to crystal negative mutation strain BMB171 by E. coli-Bt shuttle vector pHT304. A 54kDa protein with similarity to strain YBT-1518 was detected in recombinant strain, and rice shaped crystal was detected with transmission electron microscope. Bioassay indicated the LC₅₀ of recombinant strain against second lavae juvenile of Meloidgyne hapla is 9.47μg/mL, nearly equal to the original strain YBT-1518 (10.74μg/mL).

Abstract:Deep sea sediment samples of the South Sea of China were used for isolation and biodiversity examination of hydrocarbon degrading bacterium. 48 isolates were obtained from the enrichments with hexadecane as the sole carbon sources. Among them，27 isolates were capable of degrading alkane; and 4 could produce biosurfactant significantly as determined by the surface tension measurement. 2 isolates belonging to Dietzia maris lowered water surface tension to 33 mN/m. This is the first report about D. maris in biosurfactant production. The results of polymerase chain reaction (PCR) followed by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing suggested that Bacillus was the dominant member in the hexadecane enriched communities. Isolates of Bacillus aquimaris were demonstrated to be the most predominant degraders in all 7 samples at 2 sampling sites. In addition, Sporosarcina， Halomonas and Brevibacterium were also found as one of the dominant members in some samples. Therefore，species of Bacillus might play an important role in alkane degradation in the sediments of the sampling sites.

Abstract:Deep sea sediment samples of the South Sea of China were used for isolation and biodiversity examination of hydrocarbon degrading bacterium. 48 isolates were obtained from the enrichments with hexadecane as the sole carbon sources. Among them，27 isolates were capable of degrading alkane; and 4 could produce biosurfactant significantly as determined by the surface tension measurement. 2 isolates belonging to Dietzia maris lowered water surface tension to 33 mN/m. This is the first report about D. maris in biosurfactant production. The results of polymerase chain reaction (PCR) followed by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing suggested that Bacillus was the dominant member in the hexadecane enriched communities. Isolates of Bacillus aquimaris were demonstrated to be the most predominant degraders in all 7 samples at 2 sampling sites. In addition, Sporosarcina， Halomonas and Brevibacterium were also found as one of the dominant members in some samples. Therefore，species of Bacillus might play an important role in alkane degradation in the sediments of the sampling sites.

Abstract:V. harveyi VHH haemolysin, which shows high homology to the TLH haemolysin (the identities of their deduced amino acid sequences are up to 85.6%), is a putative virulence factor to marine cultured fish. A VHH probe, which is specific to V. harveyi vhhA haemolysin gene, was used to screen EcoRⅠ digests of total DNA from 57 vibrio strains，including 26 vibrio type strains, 20 V. harveyi isolates and 11 V. parahaemolyticus isolates. As a result, 1 strong hybridisation band was detected in 13 type strains, including 2 of Vibrio alginolyticus, 2 of V. harveyi, and 1 strain each of Grimontia hollisae, V. campbellii, V. cincinnatiensis, V. fischeri, V. mimicus, V. natriegens, V. parahaemolyticus, V. proteolyticus and V. logei. Also, 1 weak band was detected in 6 type strains, including V. anguillarum, V. aestuarianus, Photobacterium damselae subsp. damselae, V. fluvialis, V. furnissii and V. vulnificus. There was not any hybridization signal in other type strains. Also, vhh/tlh was present in all isolates of V. harveyi and V. parahaemolyticus. Moreover, 3 isolates of V. harveyi，i.e. VIB 645, VIB 648 and SF1, had duplicated vhh genes. The data indicates that vhh/tlh is widespread in vibrios, especially in V. harveyi related species and V. fischeri related species. To support this conclusion, the vhh/tlh homologue genes in V. anguillarum VIB 72, V. campbellii VIB 285, V. natriegens VIB 299 and V. harveyi VIB 647 were cloned and sequenced, and the deduced amino acid sequences showed high degree of identities to VHH (67%～99%) and TLH haemolysin (69％～91%). This study will help us to identify the role of vhh/tlh haemolysin gene in the pathogenicity of vibrios.

Abstract:V. harveyi VHH haemolysin, which shows high homology to the TLH haemolysin (the identities of their deduced amino acid sequences are up to 85.6%), is a putative virulence factor to marine cultured fish. A VHH probe, which is specific to V. harveyi vhhA haemolysin gene, was used to screen EcoRⅠ digests of total DNA from 57 vibrio strains，including 26 vibrio type strains, 20 V. harveyi isolates and 11 V. parahaemolyticus isolates. As a result, 1 strong hybridisation band was detected in 13 type strains, including 2 of Vibrio alginolyticus, 2 of V. harveyi, and 1 strain each of Grimontia hollisae, V. campbellii, V. cincinnatiensis, V. fischeri, V. mimicus, V. natriegens, V. parahaemolyticus, V. proteolyticus and V. logei. Also, 1 weak band was detected in 6 type strains, including V. anguillarum, V. aestuarianus, Photobacterium damselae subsp. damselae, V. fluvialis, V. furnissii and V. vulnificus. There was not any hybridization signal in other type strains. Also, vhh/tlh was present in all isolates of V. harveyi and V. parahaemolyticus. Moreover, 3 isolates of V. harveyi，i.e. VIB 645, VIB 648 and SF1, had duplicated vhh genes. The data indicates that vhh/tlh is widespread in vibrios, especially in V. harveyi related species and V. fischeri related species. To support this conclusion, the vhh/tlh homologue genes in V. anguillarum VIB 72, V. campbellii VIB 285, V. natriegens VIB 299 and V. harveyi VIB 647 were cloned and sequenced, and the deduced amino acid sequences showed high degree of identities to VHH (67%～99%) and TLH haemolysin (69％～91%). This study will help us to identify the role of vhh/tlh haemolysin gene in the pathogenicity of vibrios.

Abstract:To isolate more unique and previously unrecognized bacteria in soil samples, the culture difference under three incubation modes was investigated by using trophic, low-nutrient broth and soil extract as growth medium. Plate count proved that the oligotrophic medium resulted in a slow growth and consecutive colony formation over the course of incubation. On the 5^th day, the most number of colony-forming unit was found on trophic LB and low-nutrient R2A, which was approximate 5 times as many as that isolated on 0.1×LB. Of the 7 media, LB broth harvested the maximum bacterial communities, and novel species could be isolated as the nutrient was diluted to appropriate extent. The DGGE patterns of oligotrophic and rich nutrient culture collection displayed low similarity, however, the bands at various lanes exhibited complementary effect. When cultivated with static flask, LB and R2A media obtained more bacterial species, which concluded most species isolated by the other five media. Under the test tube incubation mode, the most species was also found in LB medium except some appeared only on R2A and TSB. Apparent bacterial communities difference could be detected between R2A, LB and TSB media. The experiment data may contribute much to the special medium design as well as improvement of bacterial culturability by using proper medium.

Abstract:To isolate more unique and previously unrecognized bacteria in soil samples, the culture difference under three incubation modes was investigated by using trophic, low-nutrient broth and soil extract as growth medium. Plate count proved that the oligotrophic medium resulted in a slow growth and consecutive colony formation over the course of incubation. On the 5^th day, the most number of colony-forming unit was found on trophic LB and low-nutrient R2A, which was approximate 5 times as many as that isolated on 0.1×LB. Of the 7 media, LB broth harvested the maximum bacterial communities, and novel species could be isolated as the nutrient was diluted to appropriate extent. The DGGE patterns of oligotrophic and rich nutrient culture collection displayed low similarity, however, the bands at various lanes exhibited complementary effect. When cultivated with static flask, LB and R2A media obtained more bacterial species, which concluded most species isolated by the other five media. Under the test tube incubation mode, the most species was also found in LB medium except some appeared only on R2A and TSB. Apparent bacterial communities difference could be detected between R2A, LB and TSB media. The experiment data may contribute much to the special medium design as well as improvement of bacterial culturability by using proper medium.

Abstract:Scorpion is an important officinal animal, and has a high nutritional value. In this study, the culture-independent and culture-dependent methods were used to investigate the microbial diversity in the scorpion's intestine. Results based on culture-independent method showed the bacteria to be related to α, β, γ-proteobacteria. Bacteria isolated by the culture-dependent method were high G+C, gram-positive bacteria. The genera Enterobacter, Serratia and Ochrobactrum were detected by both methods. To sum up the results from the two methods, the bacteria in scorpion intestine belong to 23 genera, which are Enterobacter、Serratia、Pseudomonas、Acinetobacter、Aeromonas、Citrobacter、Pedobacter、 Delftia、Ralstonia、Ochrobactrum、Sphingomonas、Exiguobacterium、Gordonia、Nocardia、Rhodococcus、Janibacte、Kocuria、Micrococcus、Agromyces、Microbacterium、Agrococcus、Deinococcus、Ornithinimicrobium, and some uncultured species. The two methods have both advantages and shortcomings. However, when used simultaneously, they complement each other．

Abstract:Scorpion is an important officinal animal, and has a high nutritional value. In this study, the culture-independent and culture-dependent methods were used to investigate the microbial diversity in the scorpion's intestine. Results based on culture-independent method showed the bacteria to be related to α, β, γ-proteobacteria. Bacteria isolated by the culture-dependent method were high G+C, gram-positive bacteria. The genera Enterobacter, Serratia and Ochrobactrum were detected by both methods. To sum up the results from the two methods, the bacteria in scorpion intestine belong to 23 genera, which are Enterobacter、Serratia、Pseudomonas、Acinetobacter、Aeromonas、Citrobacter、Pedobacter、 Delftia、Ralstonia、Ochrobactrum、Sphingomonas、Exiguobacterium、Gordonia、Nocardia、Rhodococcus、Janibacte、Kocuria、Micrococcus、Agromyces、Microbacterium、Agrococcus、Deinococcus、Ornithinimicrobium, and some uncultured species. The two methods have both advantages and shortcomings. However, when used simultaneously, they complement each other．

Abstract:A field strain C14 of chicken infectious anemia virus (CAV) was isolated from a 14 day-old broiler flock with growth runting syndromes. Antibody reactions to inactivated vaccines to avian influenza viruses (AIV) were suppressed in SPF chickens inoculated with C14 strain CAV at 1 day-old. Also C14 strain CAV and reticuloendotheliosis Virus demonstrated a synergism in immunosuppression when chickens were infected with both virus. The viral genomic DNA was amplified by PCR in 3 overlapped fragments and PCR products were cloned into T-vector plasmid for sequencing. The sequencing results indicated that the total genome of C14 strain CAV was 2298bp, it contained 3 overlapped ORF and 1 non-coding regulation fragment. Its whole genome had 97.2%-99.2% of homogeneity to other several published CAV reference strains. Sequence data indicated that there are many motifs in the non-coding area of about 400bp as the binding sites for transcriptional factors. All these motifs were very conservative. There were some mutations in 3 genes VP1, VP2 and VP3. Relatively, VP1 was less conservative than VP2 and VP3. Among different strains, mutations in these 3 genes were not correlated.

Abstract:A field strain C14 of chicken infectious anemia virus (CAV) was isolated from a 14 day-old broiler flock with growth runting syndromes. Antibody reactions to inactivated vaccines to avian influenza viruses (AIV) were suppressed in SPF chickens inoculated with C14 strain CAV at 1 day-old. Also C14 strain CAV and reticuloendotheliosis Virus demonstrated a synergism in immunosuppression when chickens were infected with both virus. The viral genomic DNA was amplified by PCR in 3 overlapped fragments and PCR products were cloned into T-vector plasmid for sequencing. The sequencing results indicated that the total genome of C14 strain CAV was 2298bp, it contained 3 overlapped ORF and 1 non-coding regulation fragment. Its whole genome had 97.2%-99.2% of homogeneity to other several published CAV reference strains. Sequence data indicated that there are many motifs in the non-coding area of about 400bp as the binding sites for transcriptional factors. All these motifs were very conservative. There were some mutations in 3 genes VP1, VP2 and VP3. Relatively, VP1 was less conservative than VP2 and VP3. Among different strains, mutations in these 3 genes were not correlated.

Abstract:Designed to investigate the potential pathogenicity of Mycoplasma genitalium (M. genitalium) and its molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression in human monocytic cells (THP-1) stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. genitalium. THP-1 cells were stimulated with LAMPs to analyze the production of proinflammatory cytokines and the expression of mRNA was detected by RT-PCR. Cell apoptosis was detected in THP-1 cells by Annexin V-propidium iodide staining. The activity of transcriptional factors, NF-κB, was examined in THP-1 cells treated with LAMPs by EMSA. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of proinflammatory cytokines, the expression of mRNA and apoptosis were also examined in THP-1 cells treated with LAMPs. M.genitalium LAMPs stimulate THP-1 cells to produce TNF-α、IL-1β and IL-6 in dose- and time-dependent manner. The mRNA levels and cell apoptosis are also downregulated in response to LAMPs stimulation and inhibited by PDTC treatment. M.genitalium LAMPs are found to trigger NF-κB activation, a possible mechanism for the induction of mRNA expression and the cell apoptosis. This study demonstrated that M.genitalium may be an important etiological factor of certain disease due to the ability of LAMPs to stimulated the expression of mRNA and apoptosis, which is probably mediated through the activation of NF-κB.

Abstract:Designed to investigate the potential pathogenicity of Mycoplasma genitalium (M. genitalium) and its molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression in human monocytic cells (THP-1) stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. genitalium. THP-1 cells were stimulated with LAMPs to analyze the production of proinflammatory cytokines and the expression of mRNA was detected by RT-PCR. Cell apoptosis was detected in THP-1 cells by Annexin V-propidium iodide staining. The activity of transcriptional factors, NF-κB, was examined in THP-1 cells treated with LAMPs by EMSA. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of proinflammatory cytokines, the expression of mRNA and apoptosis were also examined in THP-1 cells treated with LAMPs. M.genitalium LAMPs stimulate THP-1 cells to produce TNF-α、IL-1β and IL-6 in dose- and time-dependent manner. The mRNA levels and cell apoptosis are also downregulated in response to LAMPs stimulation and inhibited by PDTC treatment. M.genitalium LAMPs are found to trigger NF-κB activation, a possible mechanism for the induction of mRNA expression and the cell apoptosis. This study demonstrated that M.genitalium may be an important etiological factor of certain disease due to the ability of LAMPs to stimulated the expression of mRNA and apoptosis, which is probably mediated through the activation of NF-κB.

Abstract:The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, copper chromic salt qualitative test and RP-HPLC determination, 3 strains, l-ephedrine and d-pseudoephedrine producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO₃ as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87mg/L and d-pseudoephedrine 4.11mg/L excellular, d-pseudoephedrine 294.86mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.

Abstract:The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, copper chromic salt qualitative test and RP-HPLC determination, 3 strains, l-ephedrine and d-pseudoephedrine producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO₃ as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87mg/L and d-pseudoephedrine 4.11mg/L excellular, d-pseudoephedrine 294.86mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.

Abstract:A siderophores-producing strain E1 was isolated from the rhizosphere of cotton. Its 16S rDNA is identical to that of Pseudomonas mosselii sp.nov. at 100% level. The suicide plasmid pRL1063a carrying Tn5-1063 was introduced into E1 by triparental mating and 1000 transposon insertion mutants were subsequently screened using CAS assay. One mutant deficiency in siderophores production was obtained, namely, E1-185. DNA sequences flanking Tn5-1063 of E1-185 was amplified by TAIL-PCR. According to the DNA sequencing results, it is found that Tn5-1063 was inserted into cysI gene. The cysI of E1 is identical to that of Pseudomonas entomophila. L48 at 96% level, and similarity of amino acid sequences of their CysI is 97%. The cysI gene is required for the synthesis of cysteine. However，The ability in siderophores production of E1-185 on the plate of CAS with cysteine was recovered. It is indicated that cysI play an important role during the synthesis of siderophores. It was supposed that cysI is involved in the synthesis of acyl-S-PCPs，which is the key protein in the synthesis pathway of siderophores.

Abstract:A siderophores-producing strain E1 was isolated from the rhizosphere of cotton. Its 16S rDNA is identical to that of Pseudomonas mosselii sp.nov. at 100% level. The suicide plasmid pRL1063a carrying Tn5-1063 was introduced into E1 by triparental mating and 1000 transposon insertion mutants were subsequently screened using CAS assay. One mutant deficiency in siderophores production was obtained, namely, E1-185. DNA sequences flanking Tn5-1063 of E1-185 was amplified by TAIL-PCR. According to the DNA sequencing results, it is found that Tn5-1063 was inserted into cysI gene. The cysI of E1 is identical to that of Pseudomonas entomophila. L48 at 96% level, and similarity of amino acid sequences of their CysI is 97%. The cysI gene is required for the synthesis of cysteine. However，The ability in siderophores production of E1-185 on the plate of CAS with cysteine was recovered. It is indicated that cysI play an important role during the synthesis of siderophores. It was supposed that cysI is involved in the synthesis of acyl-S-PCPs，which is the key protein in the synthesis pathway of siderophores.

Abstract:Acetobacter xylinum NUST4.2 has been applied in the studies to examine the production, structure and thermal property of bacterial cellulose (BC) produced in stationary culture and in the stirred tank reactor. These differences are as follows: BC yield reached 7.5g/L in stationary culture for 6 days and its productivity was 0.052g/L/h. BC production reached 3.13g/L in the stirred tank reactor for 72h and its productivity was 0.043g/L/h. SEM showed that there was almost no difference between network structure built of entangled cellulose ribbons produced in static culture and in the reactor. But the cellulose ribbons produced in static culture were a much more entangled and denser network with curved and overlapping cellulose ribbons in comparison with that one produced in the stirred tank reactor. Also the thickness of the cellulose ribbons seems to differ between the two BC samples, with the one from static culture distinguished by the slightly thinner ribbons. FT-IR revealed that there was no effect of stirring on the chemical structure of BC, but intermolecular hydrogen bond of cellulose was weakened. Furthermore, BC synthesized in static culture displayed I_α-rich cellulose. XRD results indicated that no remarkable change in the cellulose crystallographic form of the BC samples. Nevertheless, BC produced in static culture was characterized by a higher crystallinity, higher I_αcontent and higher crystalline size than cellulose that was produced in the reactor. All of these results revealed that stirring in the reactor interfere strongly in the process of nascent microfibrils crystallization, favoring the formation of smaller size microfibrils and increased I_β, the more stable allomorph. Compared with cotton cellulose, the changes of thermal decomposition behavior in the BC samples were that BC produced in static culture displayed better thermal stability, but BC produced in the stirred reactor displayed better flame retarding.

Abstract:Acetobacter xylinum NUST4.2 has been applied in the studies to examine the production, structure and thermal property of bacterial cellulose (BC) produced in stationary culture and in the stirred tank reactor. These differences are as follows: BC yield reached 7.5g/L in stationary culture for 6 days and its productivity was 0.052g/L/h. BC production reached 3.13g/L in the stirred tank reactor for 72h and its productivity was 0.043g/L/h. SEM showed that there was almost no difference between network structure built of entangled cellulose ribbons produced in static culture and in the reactor. But the cellulose ribbons produced in static culture were a much more entangled and denser network with curved and overlapping cellulose ribbons in comparison with that one produced in the stirred tank reactor. Also the thickness of the cellulose ribbons seems to differ between the two BC samples, with the one from static culture distinguished by the slightly thinner ribbons. FT-IR revealed that there was no effect of stirring on the chemical structure of BC, but intermolecular hydrogen bond of cellulose was weakened. Furthermore, BC synthesized in static culture displayed I_α-rich cellulose. XRD results indicated that no remarkable change in the cellulose crystallographic form of the BC samples. Nevertheless, BC produced in static culture was characterized by a higher crystallinity, higher I_αcontent and higher crystalline size than cellulose that was produced in the reactor. All of these results revealed that stirring in the reactor interfere strongly in the process of nascent microfibrils crystallization, favoring the formation of smaller size microfibrils and increased I_β, the more stable allomorph. Compared with cotton cellulose, the changes of thermal decomposition behavior in the BC samples were that BC produced in static culture displayed better thermal stability, but BC produced in the stirred reactor displayed better flame retarding.

Abstract:Since avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar challenges when establishing infection in the extra-intestinal locations of the hosts, they may share a similar content of virulence genes and capacity to cause disease. One APEC and one UPEC isolates were compared by their content of virulence genes and other traits. The two strains showed overlap in terms of their virulence genotypes, including their possession of certain genes associated with a large transmissible plasmid of APEC,and also shared some biochemical activities. Study of the pathogenicity of UPEC in chicks showed the similar symptoms and lesions compare to those caused by APEC. Based on these results, the potential whether APEC might serve as a reservoir of plasmid-linked and virulence genes for UPEC should be considered.

Abstract:Since avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar challenges when establishing infection in the extra-intestinal locations of the hosts, they may share a similar content of virulence genes and capacity to cause disease. One APEC and one UPEC isolates were compared by their content of virulence genes and other traits. The two strains showed overlap in terms of their virulence genotypes, including their possession of certain genes associated with a large transmissible plasmid of APEC,and also shared some biochemical activities. Study of the pathogenicity of UPEC in chicks showed the similar symptoms and lesions compare to those caused by APEC. Based on these results, the potential whether APEC might serve as a reservoir of plasmid-linked and virulence genes for UPEC should be considered.

Abstract:Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia. Apx toxin, an exotoxin secreted by A. pleuropneumoniae, is one of the most important virulence factors. To construct an avirulent mutant strain by inactivation of ApxI toxin, the apxIC gene of A. pleuropneumoniae serovar 10 was inactivated by inserting a chloramphenicol resistance gene cassette into the downstream XhoI site of the apxIC gene for constructing the transfer plasmid. The transfer plasmid was introduced into the electrocompetent A. pleuropneumoniae serovar 10 for homologous recombination by electroporation. The mutant strain was obtained and identified by PCR and Southern blotting. The mutant strain was phenotypically identical to the parent strain except that it showed no haemolytic activity. The mutant strain was also able to secret the same ApxI toxin as the parent strain. In the intra-peritoneal mouse model, the virulence of the mutant strain decreased at least 100 fold compared with the parent strain. The mutant was evaluated as a potential vaccine using a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant with 14 days' interval and then challenged 14 days after the last vaccination with A. pleuropneumoniae serovar 1 and serovar 10 reference strains respectively. The death number and lung lesion score in the vaccinated pigs given the serovar 1 challenge were obviously lower than those in the unvaccinated pigs. And the lower lung lesion score was also observed in the vaccinated pigs challenged with serovar 10. And the positive numbers of A. pleuropneumoniae re-isolation and PCR detection showed the same consistency. The vaccination-challenge trial suggested that the mutant strain could offer partial cross-protection as a live attenuated vaccine against A. pleuropneumoniae infection.

Abstract:Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia. Apx toxin, an exotoxin secreted by A. pleuropneumoniae, is one of the most important virulence factors. To construct an avirulent mutant strain by inactivation of ApxI toxin, the apxIC gene of A. pleuropneumoniae serovar 10 was inactivated by inserting a chloramphenicol resistance gene cassette into the downstream XhoI site of the apxIC gene for constructing the transfer plasmid. The transfer plasmid was introduced into the electrocompetent A. pleuropneumoniae serovar 10 for homologous recombination by electroporation. The mutant strain was obtained and identified by PCR and Southern blotting. The mutant strain was phenotypically identical to the parent strain except that it showed no haemolytic activity. The mutant strain was also able to secret the same ApxI toxin as the parent strain. In the intra-peritoneal mouse model, the virulence of the mutant strain decreased at least 100 fold compared with the parent strain. The mutant was evaluated as a potential vaccine using a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant with 14 days' interval and then challenged 14 days after the last vaccination with A. pleuropneumoniae serovar 1 and serovar 10 reference strains respectively. The death number and lung lesion score in the vaccinated pigs given the serovar 1 challenge were obviously lower than those in the unvaccinated pigs. And the lower lung lesion score was also observed in the vaccinated pigs challenged with serovar 10. And the positive numbers of A. pleuropneumoniae re-isolation and PCR detection showed the same consistency. The vaccination-challenge trial suggested that the mutant strain could offer partial cross-protection as a live attenuated vaccine against A. pleuropneumoniae infection.

Abstract:Three hybridoma cell lines, 2C6, 5B7 and 12G9, secreting monoclonal antibodies (McAbs) against Cymbidium mosaic virus (CymMV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the CymMV particles. The three McAbs could specifically react with CymMV. The titres of ascitic fluids of two McAbs are up to 10-6 in I-ELISA. Isotypes and subclasses of the the three McAbs belong to IgG1. Isotypes of light strains of the three McAbs all belong to κ. They were used in antigen-coated plate (ACP) -ELISA for CymMV detection, and ACP-ELISA could successfully detect 0.487ng of purified CymMV or virus in plant sap diluted 1∶10240. The presence of CymMV in field Orchids tissues was investigated with ACP-ELISA.

Abstract:Three hybridoma cell lines, 2C6, 5B7 and 12G9, secreting monoclonal antibodies (McAbs) against Cymbidium mosaic virus (CymMV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the CymMV particles. The three McAbs could specifically react with CymMV. The titres of ascitic fluids of two McAbs are up to 10-6 in I-ELISA. Isotypes and subclasses of the the three McAbs belong to IgG1. Isotypes of light strains of the three McAbs all belong to κ. They were used in antigen-coated plate (ACP) -ELISA for CymMV detection, and ACP-ELISA could successfully detect 0.487ng of purified CymMV or virus in plant sap diluted 1∶10240. The presence of CymMV in field Orchids tissues was investigated with ACP-ELISA.

Abstract:Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis， whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

Abstract:Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis， whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

Abstract:Moderately halophilic bacteria which grow best in media with 3% to 15% salt constitute a heterogenous group of microorganisms which belong to different genera. These bacteria can inhabit the salt or soda lakes, coastal lagoons or man-made salterns. Moderately halophilc bacteria living in higher saline environments can not only cope with high osmotic stress but also adapt osmotic shock in short time. To adapt to these environments, all the species make a osmoprotection by the accumulation a restricted range of low molecular mass molecules, small, organic compatible solutes, such as sugars, amino acids, betaines and ectoines. Therefore, the osmoadaptation of moderately halophilc bacteria is regulated by the so-called “compatible solute" strategy. Compatible solutes are operationally defined as organic osmolytes that can be amassed by the cell in exceedingly high concentrations without disturbing vital cellular functions and the correct folding of proteins. As a result, compatible solutes can make important contributions to the restoration of the turgor under conditions of low water activity by counteracting the efflux of water from the cell. In addition, they have a stabilizing, both in vivo and vitro, on the native structure of proteins and cell components. This mechanism has a minimal requirement for genetic change and a high degree of flexibility in allowing moderate halophiles to adapt to saline environment. In this review, the adaptation to saline environments, the variety and characteristic of compatible solutes, and the functional mechanism of moderately halophilic bacteria are reviewed and discussed.

Abstract:Moderately halophilic bacteria which grow best in media with 3% to 15% salt constitute a heterogenous group of microorganisms which belong to different genera. These bacteria can inhabit the salt or soda lakes, coastal lagoons or man-made salterns. Moderately halophilc bacteria living in higher saline environments can not only cope with high osmotic stress but also adapt osmotic shock in short time. To adapt to these environments, all the species make a osmoprotection by the accumulation a restricted range of low molecular mass molecules, small, organic compatible solutes, such as sugars, amino acids, betaines and ectoines. Therefore, the osmoadaptation of moderately halophilc bacteria is regulated by the so-called “compatible solute" strategy. Compatible solutes are operationally defined as organic osmolytes that can be amassed by the cell in exceedingly high concentrations without disturbing vital cellular functions and the correct folding of proteins. As a result, compatible solutes can make important contributions to the restoration of the turgor under conditions of low water activity by counteracting the efflux of water from the cell. In addition, they have a stabilizing, both in vivo and vitro, on the native structure of proteins and cell components. This mechanism has a minimal requirement for genetic change and a high degree of flexibility in allowing moderate halophiles to adapt to saline environment. In this review, the adaptation to saline environments, the variety and characteristic of compatible solutes, and the functional mechanism of moderately halophilic bacteria are reviewed and discussed.

Abstract:Noroviruses (NVs) were one of the new borne viruses, which was found firstly in the Unit States of America in 1972 and reported in China in 1995. The main food-borne viral pathogens affect people badly and cause the epidemic acute gastroenteritis in all kinds of people. And to this day, however, no cell lines and animal models have been found, which has hampered the study of these viruses. With the progress of the molecular biology and other subjects, the genomes of different NVs were sequenced, and the proteins of the viruses were expressed in vitro by the eukaryotic and prokaryotic expression systems respectively. Therefore, the novel knowledge and ideas on NVs were developed quickly in the character of these kinds of viruses. In this article, the NVs were described systematically, such as the structure of genomes and function of these nucleotides, organization and function of proteins, application and development of detecting and accumulation of epidemiology. Furthermore, the majority of researchers were interested in and focused on the study of protein and the detection of viruses. The progress and obstacles in this field were also involved. In additional, the suggestions were mentioned about the molecular evolution, detection and multiplication system in vitro on the viruses.

Abstract:Noroviruses (NVs) were one of the new borne viruses, which was found firstly in the Unit States of America in 1972 and reported in China in 1995. The main food-borne viral pathogens affect people badly and cause the epidemic acute gastroenteritis in all kinds of people. And to this day, however, no cell lines and animal models have been found, which has hampered the study of these viruses. With the progress of the molecular biology and other subjects, the genomes of different NVs were sequenced, and the proteins of the viruses were expressed in vitro by the eukaryotic and prokaryotic expression systems respectively. Therefore, the novel knowledge and ideas on NVs were developed quickly in the character of these kinds of viruses. In this article, the NVs were described systematically, such as the structure of genomes and function of these nucleotides, organization and function of proteins, application and development of detecting and accumulation of epidemiology. Furthermore, the majority of researchers were interested in and focused on the study of protein and the detection of viruses. The progress and obstacles in this field were also involved. In additional, the suggestions were mentioned about the molecular evolution, detection and multiplication system in vitro on the viruses.