Abstract:Bacterial biodiversities of three moderately thermophilic bioleaching microfloras grown at 50℃ on media with pyrite, chalcopyrite, and pure ferrous iron supplemented with sulfur as energy sources were investigated respectively. The 16S rRNA genes of the microorganisms in the cultures flasks were PCR amplified and cloned to identify the bacterial species by comparative sequence analysis, the structural differences of microfloras enriched by different energy sources were compared. A total of 303 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. Cluster analysis identified 29 unique RFLP patterns, and the inserted 16S rRNA genes sequences were determined and for phylogenetic analysis. Most of sequences obtained were similar (89.1%～99.7%) to the 16S rRNA gene sequences of the reported bioleaching microorganisms. The species identified from the flasks during bioleaching of pyrite, pure ferrous iron supplemented with sulfur, and chalcopyrite were closely related to Acidithiobacillus caldus, Sulfobacillus thermotolerans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferriphilum, two uncultured forest soil bacterium clones and one uncultured proteobacterium clone. Among these bacteria, Acidithiobacillus caldus, Sulfobacillus thermotolerans and Leptospirillum ferriphilum were the dominant bacterial species. L. ferriphilum was the most dominant species in microfloras enriched in media with pyrite and ferrous iron supplemented with sulfur as energy sources, the abundance were 53.8% and 45.9% respectively. In the culture with chalcopyrite as energy sources, S. thermotolerans had the highest abundance of 70.1%.

Abstract:Bacterial biodiversities of three moderately thermophilic bioleaching microfloras grown at 50℃ on media with pyrite, chalcopyrite, and pure ferrous iron supplemented with sulfur as energy sources were investigated respectively. The 16S rRNA genes of the microorganisms in the cultures flasks were PCR amplified and cloned to identify the bacterial species by comparative sequence analysis, the structural differences of microfloras enriched by different energy sources were compared. A total of 303 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. Cluster analysis identified 29 unique RFLP patterns, and the inserted 16S rRNA genes sequences were determined and for phylogenetic analysis. Most of sequences obtained were similar (89.1%～99.7%) to the 16S rRNA gene sequences of the reported bioleaching microorganisms. The species identified from the flasks during bioleaching of pyrite, pure ferrous iron supplemented with sulfur, and chalcopyrite were closely related to Acidithiobacillus caldus, Sulfobacillus thermotolerans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferriphilum, two uncultured forest soil bacterium clones and one uncultured proteobacterium clone. Among these bacteria, Acidithiobacillus caldus, Sulfobacillus thermotolerans and Leptospirillum ferriphilum were the dominant bacterial species. L. ferriphilum was the most dominant species in microfloras enriched in media with pyrite and ferrous iron supplemented with sulfur as energy sources, the abundance were 53.8% and 45.9% respectively. In the culture with chalcopyrite as energy sources, S. thermotolerans had the highest abundance of 70.1%.

Abstract:Catechol 1,2-dioxygenase gene, catA, from naphthalene-degrading plasmid pND6-1 of Pseudomonas putida ND6, was cloned and expressed in Escherichia coli. Enzymic properties of the expressed product were investigated. The results indicated that the K_m and V_max of the enzyme are 0.019 mol/L and 1.434 mol/(min·mg), respectively. The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50℃ for 45min. Fe²⁺ could enhance the enzyme activity by 292%. The enzyme displayed a lower activity against 4-chlorocatechol and belongs to group Ⅰ of catechol 1,2-dioxygenases. When naphthalene was used as a substrate for growth of strain ND6， catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were both detected in their crude extract. However, when strain ND6 was grown on benzoate, ρ-hydroxybenzoic acid or phenylacetic acid as a sole source of carbon the activity of catechol 1,2-dioxygenase was much higher than that of catechol 2,3-dioxygenase. These indicated that strain ND6 is able to metabolize naphthalene by catechol meta- and ortho-cleavage pathways. When benzoate, ρ-hydroxybenzoic acid and phenylacetic acid were used as growth substrates, strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.

Abstract:Catechol 1,2-dioxygenase gene, catA, from naphthalene-degrading plasmid pND6-1 of Pseudomonas putida ND6, was cloned and expressed in Escherichia coli. Enzymic properties of the expressed product were investigated. The results indicated that the K_m and V_max of the enzyme are 0.019 mol/L and 1.434 mol/(min·mg), respectively. The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50℃ for 45min. Fe²⁺ could enhance the enzyme activity by 292%. The enzyme displayed a lower activity against 4-chlorocatechol and belongs to group Ⅰ of catechol 1,2-dioxygenases. When naphthalene was used as a substrate for growth of strain ND6， catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were both detected in their crude extract. However, when strain ND6 was grown on benzoate, ρ-hydroxybenzoic acid or phenylacetic acid as a sole source of carbon the activity of catechol 1,2-dioxygenase was much higher than that of catechol 2,3-dioxygenase. These indicated that strain ND6 is able to metabolize naphthalene by catechol meta- and ortho-cleavage pathways. When benzoate, ρ-hydroxybenzoic acid and phenylacetic acid were used as growth substrates, strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.

Abstract:Bacillus thuringiensis is a member of the B. cereus group, which also contains B. cereus, B. mycoides, B. pseudomycoides, B. anthracis and B. weihenstephanensis. Among them, B. thuringiensis and B. cereus share a high level chromosomal similarity and are phenotypically similar except that B. thuringiensis has insecticidal plasmid encoding crystal proteins. Twenty-six B. cereus group strains were surveyed in this study for the presence of enterotoxin genes and other toxin genes related to pathogenicity. PCR results showed that the pleiotropic virulence regulator plcR was presented in 17 B. cereus group strains. About 73% of the B. cereus group strains and 83% of the B. thuringiensis strains contained at least one of the three hbl genes and one of the three nhe genes, indicating that B. thuringiensis, including strains used commercially, had enterotoxin encoding genes. Additionally, B. cereus DBt248 was proved to be devoid of all three hbl genes, three nhe genes or plcR. Thus this strain might be a potential candidate as a host strain for expressing B. thuringiensis crystal toxins to construct safety insecticidal engineering strains without enterotoxic activity.

Abstract:Bacillus thuringiensis is a member of the B. cereus group, which also contains B. cereus, B. mycoides, B. pseudomycoides, B. anthracis and B. weihenstephanensis. Among them, B. thuringiensis and B. cereus share a high level chromosomal similarity and are phenotypically similar except that B. thuringiensis has insecticidal plasmid encoding crystal proteins. Twenty-six B. cereus group strains were surveyed in this study for the presence of enterotoxin genes and other toxin genes related to pathogenicity. PCR results showed that the pleiotropic virulence regulator plcR was presented in 17 B. cereus group strains. About 73% of the B. cereus group strains and 83% of the B. thuringiensis strains contained at least one of the three hbl genes and one of the three nhe genes, indicating that B. thuringiensis, including strains used commercially, had enterotoxin encoding genes. Additionally, B. cereus DBt248 was proved to be devoid of all three hbl genes, three nhe genes or plcR. Thus this strain might be a potential candidate as a host strain for expressing B. thuringiensis crystal toxins to construct safety insecticidal engineering strains without enterotoxic activity.

Abstract:The hrp genes of Xanthomonas oryzae pv.oryzicola (Xooc), which is the causal agent of bacterial leaf streak in rice, possesses the ability to elicit hypersensitive response on nonhost plants and the pathogenicity in host rice. In order to analyze the function of the hrp genes, we developed hrp-inducing systems using transcriptional hrp::gfp fusions with the promoters of hrpX and hpa1 of Xooc. The levels of GFP protein expression indicated that the hrp gene expression in Xooc was not efficiently induced in NB medium, but efficiently in XOM3 medium. Using the hrpG and hrpX mutants of Xooc as the controls, the results by RT-PCR demonstrated that in wild type strain the expression of the hpa1 gene was suppressed in NB medium, but was increased in XOM3 medium. When incubated in XOM3, the expression of the hpa1 gene was abolished in hrpX mutant, while the level of the hpa1 gene expression was lower in the hrpG mutant than that in wild-type strain. More importantly, it was found that the induction of the hrp gene expression was strongly increased in response to rice suspension cells and callus in this study. This suggests that the hrp-inducing systems, XOM3 or rice suspension cells or rice callus, for the induction of the hrp genes expression be useful for functionally analyzing the hrp genes, mining effectors secreted by the type Ⅲ secretion apparatus and understanding pathogenicity determinats of Xooc.

Abstract:The hrp genes of Xanthomonas oryzae pv.oryzicola (Xooc), which is the causal agent of bacterial leaf streak in rice, possesses the ability to elicit hypersensitive response on nonhost plants and the pathogenicity in host rice. In order to analyze the function of the hrp genes, we developed hrp-inducing systems using transcriptional hrp::gfp fusions with the promoters of hrpX and hpa1 of Xooc. The levels of GFP protein expression indicated that the hrp gene expression in Xooc was not efficiently induced in NB medium, but efficiently in XOM3 medium. Using the hrpG and hrpX mutants of Xooc as the controls, the results by RT-PCR demonstrated that in wild type strain the expression of the hpa1 gene was suppressed in NB medium, but was increased in XOM3 medium. When incubated in XOM3, the expression of the hpa1 gene was abolished in hrpX mutant, while the level of the hpa1 gene expression was lower in the hrpG mutant than that in wild-type strain. More importantly, it was found that the induction of the hrp gene expression was strongly increased in response to rice suspension cells and callus in this study. This suggests that the hrp-inducing systems, XOM3 or rice suspension cells or rice callus, for the induction of the hrp genes expression be useful for functionally analyzing the hrp genes, mining effectors secreted by the type Ⅲ secretion apparatus and understanding pathogenicity determinats of Xooc.

Abstract:Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon (EZ::TN) insertional mutant library of Xoo PXO99 against a host plant (rice cultivar IR24), one virulence-deficient mutant, XOG11, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction (TAIL-PCR) and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type Ⅲ secretion system (TTSS) and locates at the downstream of hrpE. The product of hpaB in Xoo is a small (Molecular Weight, 17.6kDa), acidic (PI, 4.28) and Leucine-rich (14.4%) protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TTSS chaperone. Mutant XOG11 was confirmed both by PCR and Southern blotting: The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOG11 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOG11 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHM1 and transferring the recombinant plasmid into XOG11 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOG11 is due to the mutation of hpaB gene.

Abstract:Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon (EZ::TN) insertional mutant library of Xoo PXO99 against a host plant (rice cultivar IR24), one virulence-deficient mutant, XOG11, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction (TAIL-PCR) and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type Ⅲ secretion system (TTSS) and locates at the downstream of hrpE. The product of hpaB in Xoo is a small (Molecular Weight, 17.6kDa), acidic (PI, 4.28) and Leucine-rich (14.4%) protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TTSS chaperone. Mutant XOG11 was confirmed both by PCR and Southern blotting: The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOG11 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOG11 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHM1 and transferring the recombinant plasmid into XOG11 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOG11 is due to the mutation of hpaB gene.

Abstract:Direct screening of bacterial genes expressed during infection in the host is limited, because isolation of bacterial transcripts from host tissues necessitates separation from the abundance of host RNA. Selective capture of transcribed sequences (SCOTS) allows the selective capture of bacterial cDNA derived from infected tissues using hybridization to biotinylated bacterial genomic DNA. Avian pathogenic E.coli strain E037 (serogroup O78) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. Three-week-old white leghorn specific-pathogen-free chickens were inoculated into the right thoracic air sac with a 0.1mL suspension containing 107CFU of APEC strain E037. Total RNA was isolated from infected tissues (pericardium and air sacs) 6 or 24h postinfection and converted to cDNAs. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-pathogenic E.coli K-12 strain ) transcripts, pathogen-specific cDNAs were identified. Randomly chosen cDNA clones derived from transcripts in the air sacs or pericardium were selected and sequenced. The clones, termed aec, contained numerous APEC-specific sequences. Among the distinct 31 aec clones, pathogen-specific clones contained sequences homologous to known and novel putative bacterial virulence gene products involved in adherence, iron transport, lipopolysaccharide (LPS) synthesis, plasmid replication and conjugation, putative phage encoded products, and gene products of unknown function. Overall, the current study provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression of a pathogenic E.coli strain in a natural animal host during the infectious process.

Abstract:Direct screening of bacterial genes expressed during infection in the host is limited, because isolation of bacterial transcripts from host tissues necessitates separation from the abundance of host RNA. Selective capture of transcribed sequences (SCOTS) allows the selective capture of bacterial cDNA derived from infected tissues using hybridization to biotinylated bacterial genomic DNA. Avian pathogenic E.coli strain E037 (serogroup O78) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. Three-week-old white leghorn specific-pathogen-free chickens were inoculated into the right thoracic air sac with a 0.1mL suspension containing 107CFU of APEC strain E037. Total RNA was isolated from infected tissues (pericardium and air sacs) 6 or 24h postinfection and converted to cDNAs. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-pathogenic E.coli K-12 strain ) transcripts, pathogen-specific cDNAs were identified. Randomly chosen cDNA clones derived from transcripts in the air sacs or pericardium were selected and sequenced. The clones, termed aec, contained numerous APEC-specific sequences. Among the distinct 31 aec clones, pathogen-specific clones contained sequences homologous to known and novel putative bacterial virulence gene products involved in adherence, iron transport, lipopolysaccharide (LPS) synthesis, plasmid replication and conjugation, putative phage encoded products, and gene products of unknown function. Overall, the current study provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression of a pathogenic E.coli strain in a natural animal host during the infectious process.

Abstract:Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1×10⁶cfu/mL, total clones of 1.2×10⁷cfu and an average insertion size of about 2243bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5×10⁵cfu/mL, total clones of 5.5×10⁶cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

Abstract:Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1×10⁶cfu/mL, total clones of 1.2×10⁷cfu and an average insertion size of about 2243bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5×10⁵cfu/mL, total clones of 5.5×10⁶cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

Abstract:NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine,such as IFN,TNF,etc. A motif binding to CBSF，conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain,is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result，the work pays the way for further understanding the role of NS1 in human H5N1 infection and development of new antiviral drugs against influenza virus.

Abstract:NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine,such as IFN,TNF,etc. A motif binding to CBSF，conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain,is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result，the work pays the way for further understanding the role of NS1 in human H5N1 infection and development of new antiviral drugs against influenza virus.

Abstract:Six recombinant plasmids covering cDNA of porcine reproductive and respiratory syndrome virus BJ-4 were sequenced, respectively, and 23 point mutations were reverted with site-directed mutagenesis kit. The full-length cDNA clone pWSK-DCBA was assembled and re-sequenced. The capped viral genomic RNA was transcribed in vitro, mixed with liposome and transfected into MARC-145 cells, and an infectious virus (designated rV68) was rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 and stably propagated in vitro. Growth kinetics curve of the rV68 exhibited a delayed replication in MARC-145 cell, namely its peak titer time was 12h later than that of parental virus. However, there was no significant difference between the peak titers of the rescued and parental virus (P＞0.05). These results suggest that the full-length cDNA clone pWSK-DCBA of PRRSV BJ-4 is infectious, which provide a basis for further study on molecular pathogenicity and immunity, as well as developing novel vaccine of PRRSV.

Abstract:Six recombinant plasmids covering cDNA of porcine reproductive and respiratory syndrome virus BJ-4 were sequenced, respectively, and 23 point mutations were reverted with site-directed mutagenesis kit. The full-length cDNA clone pWSK-DCBA was assembled and re-sequenced. The capped viral genomic RNA was transcribed in vitro, mixed with liposome and transfected into MARC-145 cells, and an infectious virus (designated rV68) was rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 and stably propagated in vitro. Growth kinetics curve of the rV68 exhibited a delayed replication in MARC-145 cell, namely its peak titer time was 12h later than that of parental virus. However, there was no significant difference between the peak titers of the rescued and parental virus (P＞0.05). These results suggest that the full-length cDNA clone pWSK-DCBA of PRRSV BJ-4 is infectious, which provide a basis for further study on molecular pathogenicity and immunity, as well as developing novel vaccine of PRRSV.

Abstract:Batch cultures of Escherichia coli DH5α and DA19 in carbon source limited defined media and continuous cultures in nitrogen source limited defined media were performed to compare the metabolism in both strains. DH5α showed improved growth and less acetic acid production after supplementation of adenine, but adenine had little effects on the metabolism of DA19. The proteomic analysis showed that expression of purH was down regulated in DA19 by addition of adenine while was unchanged in DH5α, suggesting that the de novo synthesis ability of purine nucleotide in DH5α was poorer than that in DA19. Supplementation of adenine resulted in enhanced expression of gnd and yddS in DH5α, and thus improved the growth.

Abstract:Batch cultures of Escherichia coli DH5α and DA19 in carbon source limited defined media and continuous cultures in nitrogen source limited defined media were performed to compare the metabolism in both strains. DH5α showed improved growth and less acetic acid production after supplementation of adenine, but adenine had little effects on the metabolism of DA19. The proteomic analysis showed that expression of purH was down regulated in DA19 by addition of adenine while was unchanged in DH5α, suggesting that the de novo synthesis ability of purine nucleotide in DH5α was poorer than that in DA19. Supplementation of adenine resulted in enhanced expression of gnd and yddS in DH5α, and thus improved the growth.

Abstract:The distal mammalian gut harbors prodigiously abundant microbes, which provide unique metabolic traits to host. A lactate-utilizing, butyrate-producing bacterium, strain LB01, was isolated from adult swine feces by utilizing modified Hungate technique with rumen liquid-independent YCFA medium supplemented with lactate as the single carbon source. It was an obligate anaerobic, Gram positive bacterium, and could utilize glucose, fructose, maltose and lactate with a large amount of gas products. 16S rRNA sequence analysis revealed that it had the high similarity with members of the genus Megasphaera. The metabolic characteristics of strain LB01 was investigated by using in vitro fermentation system. Lactate at the concentration of 65 mmol/L in YCFA medium was rapidly consumed within 9 hours and was mainly converted to propionate and butyrate after 24h. As the level of acetate declined, the concentration of butyrate rose only in the presence of glucose, suggesting that butyrate could possibly be synthesized by the acetyl CoA: butyryl CoA transferase. When co-cultured with lactic acid bacteria strain K9, strain LB01 evidently reduced the concentration of lactate produced by strain K9 and decelerated the rapid pH drop, finally producing 12.11mmol/L butyrate and 4.06mmol/L propionate. The metabolic characteristics that strain LB01 efficiently converts toxic lactate and excessive acetate to butyrate can prevent lactate and acetate accumulation in the large intestine and maintain the slightly acidic environment of the large intestine, consequently revealing that stain LB01 could act as a potential probiotics.

Abstract:The distal mammalian gut harbors prodigiously abundant microbes, which provide unique metabolic traits to host. A lactate-utilizing, butyrate-producing bacterium, strain LB01, was isolated from adult swine feces by utilizing modified Hungate technique with rumen liquid-independent YCFA medium supplemented with lactate as the single carbon source. It was an obligate anaerobic, Gram positive bacterium, and could utilize glucose, fructose, maltose and lactate with a large amount of gas products. 16S rRNA sequence analysis revealed that it had the high similarity with members of the genus Megasphaera. The metabolic characteristics of strain LB01 was investigated by using in vitro fermentation system. Lactate at the concentration of 65 mmol/L in YCFA medium was rapidly consumed within 9 hours and was mainly converted to propionate and butyrate after 24h. As the level of acetate declined, the concentration of butyrate rose only in the presence of glucose, suggesting that butyrate could possibly be synthesized by the acetyl CoA: butyryl CoA transferase. When co-cultured with lactic acid bacteria strain K9, strain LB01 evidently reduced the concentration of lactate produced by strain K9 and decelerated the rapid pH drop, finally producing 12.11mmol/L butyrate and 4.06mmol/L propionate. The metabolic characteristics that strain LB01 efficiently converts toxic lactate and excessive acetate to butyrate can prevent lactate and acetate accumulation in the large intestine and maintain the slightly acidic environment of the large intestine, consequently revealing that stain LB01 could act as a potential probiotics.

Abstract:A new bacterium with potential biocontrol ability, Pseudomonas sp. M18, was isolated from the soil of agricultural field in suburb of Shanghai (China). It had been demonstrated that biosynthesis and secretion of phenazine-1-carboxylic acid and pyoluteorin in Pseudomonas sp. M18 contributes to its suppression of soilborne pathogens. In order to study the correlation and regulatory mechanism of two antifungal compounds biosynthesis, the mutant M18T and M18Z1 were constructed with insertion of the gentamycin resistance gene cassette (aacC1), respectively. With introduction of the translational fusion pMEAZ (pltA`-'lacZ) into the wild type strain M18 or the plt-mutant M18T, respectively, it was found that β-galactosidase activities of the mutant M18T (pMEAZ) are remarkably enhanced by adding a certain amount of pyoluteorin in KMB medium. The results indicated that pyoluteorin might positively autoinduce expression of the plt gene loci. In investigating the correlation of two antifungal agents, it was showed that the pyoluteorin-negative mutant M18T produces the same level of phenazine-1-carboxylic acid in comparison with the wild type strain M18. Overexpression of the plt gene loci does not result in decrease of phenazine-1-carboxylic acid in a pltZ-mutant of Pseudomonas sp. M18. However, the distinct decrease of phenazine-1-carboxylic acid biosynthesis does lead to enhanced biosynthesis of pyoluteorin in the mutant M18Z1. Addition of phenazine-1-carboxylic acid in KMB medium makes the mutant M18S produce less pyoluteorin. These results indicated that a special correlation of secondary antifungal agents biosynthesis seems to be existed in Pseudomonas sp. M18, i.e., production of pyoluteorin does not exert any influence on expression of the phz gene cluster, while phenazine-1-carboxylic acid makes negative impact on the biosynthesis of pyoluteorin.

Abstract:A new bacterium with potential biocontrol ability, Pseudomonas sp. M18, was isolated from the soil of agricultural field in suburb of Shanghai (China). It had been demonstrated that biosynthesis and secretion of phenazine-1-carboxylic acid and pyoluteorin in Pseudomonas sp. M18 contributes to its suppression of soilborne pathogens. In order to study the correlation and regulatory mechanism of two antifungal compounds biosynthesis, the mutant M18T and M18Z1 were constructed with insertion of the gentamycin resistance gene cassette (aacC1), respectively. With introduction of the translational fusion pMEAZ (pltA`-'lacZ) into the wild type strain M18 or the plt-mutant M18T, respectively, it was found that β-galactosidase activities of the mutant M18T (pMEAZ) are remarkably enhanced by adding a certain amount of pyoluteorin in KMB medium. The results indicated that pyoluteorin might positively autoinduce expression of the plt gene loci. In investigating the correlation of two antifungal agents, it was showed that the pyoluteorin-negative mutant M18T produces the same level of phenazine-1-carboxylic acid in comparison with the wild type strain M18. Overexpression of the plt gene loci does not result in decrease of phenazine-1-carboxylic acid in a pltZ-mutant of Pseudomonas sp. M18. However, the distinct decrease of phenazine-1-carboxylic acid biosynthesis does lead to enhanced biosynthesis of pyoluteorin in the mutant M18Z1. Addition of phenazine-1-carboxylic acid in KMB medium makes the mutant M18S produce less pyoluteorin. These results indicated that a special correlation of secondary antifungal agents biosynthesis seems to be existed in Pseudomonas sp. M18, i.e., production of pyoluteorin does not exert any influence on expression of the phz gene cluster, while phenazine-1-carboxylic acid makes negative impact on the biosynthesis of pyoluteorin.

Abstract:Terric acid，an antifungal metabolites produced by a marine mold Aspergillus flavis No.179,was isolated and purified. The effects of terric acid at different concertrations on the growth and cellular morphology of C. albicans AS2,538 were studied by the method of liquid dilution. The substance inhibits the growth of C. albicans AS2,538 obviously at low concentration and has fungicidal action at high concentration. The minimum inhibiting concentrations(MIC) and minimum lethal concentrations(MLC) on C. albicans AS2,538 were 6.25μg/mL and 50μg/mL, respectively. At Sub-MIC（3.13μg/mL）, terric acid inhibited the formation of pseudo-hyphal of C. albicans AS2,538 and caused cell morphological change. After addition of terric acid to the medium to final concentration of 50μg/mL, cell morphology of C. albicans AS2,538 was irregular and some cells turned bigger and disrupted. The effects of terric acid on the synthesis of macromolecules, DNA, RNA or proteins of C. albicans AS2,538 were estimated by measurement of incorporation of radioactive precursors,¹⁴C-Thymidine, ³H-Uridine or¹⁴C-Valine, respectively. When C. albicans AS2,538 was incubated for 4h in 5 g/mL terric acid medium, the incorporation of radioactive isoleucine and uridine decreased 47.8%, 70.7%, respectively, Compared with controls incubated in commonly medium. However, the incorporation of ［3H］thymidine did not have significant change. This results indicated that terric acid inhibits the synthesis of protein and RNA but have no effect on DNA synthesis in a C．albicans AS2,538.

Abstract:Terric acid，an antifungal metabolites produced by a marine mold Aspergillus flavis No.179,was isolated and purified. The effects of terric acid at different concertrations on the growth and cellular morphology of C. albicans AS2,538 were studied by the method of liquid dilution. The substance inhibits the growth of C. albicans AS2,538 obviously at low concentration and has fungicidal action at high concentration. The minimum inhibiting concentrations(MIC) and minimum lethal concentrations(MLC) on C. albicans AS2,538 were 6.25μg/mL and 50μg/mL, respectively. At Sub-MIC（3.13μg/mL）, terric acid inhibited the formation of pseudo-hyphal of C. albicans AS2,538 and caused cell morphological change. After addition of terric acid to the medium to final concentration of 50μg/mL, cell morphology of C. albicans AS2,538 was irregular and some cells turned bigger and disrupted. The effects of terric acid on the synthesis of macromolecules, DNA, RNA or proteins of C. albicans AS2,538 were estimated by measurement of incorporation of radioactive precursors,¹⁴C-Thymidine, ³H-Uridine or¹⁴C-Valine, respectively. When C. albicans AS2,538 was incubated for 4h in 5 g/mL terric acid medium, the incorporation of radioactive isoleucine and uridine decreased 47.8%, 70.7%, respectively, Compared with controls incubated in commonly medium. However, the incorporation of ［3H］thymidine did not have significant change. This results indicated that terric acid inhibits the synthesis of protein and RNA but have no effect on DNA synthesis in a C．albicans AS2,538.

Abstract:A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%～40% homology to those of related enzymes from various sources and 80%～90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b(+) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6×His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS_PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of >99%.The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. Ｔhe enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.

Abstract:A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%～40% homology to those of related enzymes from various sources and 80%～90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b(+) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6×His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS_PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of >99%.The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. Ｔhe enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.

Abstract:Mosquitocidal toxin 1 (Mtx1) was synthesized during vegetative phase of Bacillus sphaericus and it had been proved to have higher activity to Aedes spp. larvae and Binary toxin (Bin) resistance Culex larvae. The truncated 97kDa Mtx1 with a deletion of the signal peptide and the Cyt1Aa crystal protein, a 27.3kDa δ-endotoxin from Bacillus thuringiensis subsp. israelensis (Bti), were purified from Escherichia coli and B. thuringiensis recombinant strains respectively. Both purified toxins had high toxicity against Culex quinquefasciatus larvae. Bioassay result revealed the purified Mtx1 toxin had high toxicity against the target mosquito larvae, with LC₅₀ of 45.2ng/mL. However, the mixture of Mtx1 and Cyt1Aa exhibited higher toxicity against the mosquito larvae, with a lowest LC₅₀ value of 20.19ng/mL at the ratio of 1∶3. (Mtx1:Cyt1Aa). The calculated synergistic factor of different mixtures suggested a strong synergistic effect between Cyt1Aa toxin and Mtx1. Furthermore, the presence of Cyt1Aa in the mixture could induce early larval mortality, enhancing the activity of Mtx1 to the target mosquito larvae. The synergistic effect of Cyt1Aa on mortality of Mtx1 to mosquito larvae might be caused by the damage of the larval midgut-hemocoel barrier induced by the activated Cyt1Aa toxin, which enhanced the specific pathogenesis of Mtx1 on mosquito larvae. It is suggested that the co-application of Mtx1 and Cyt1Aa in future will be integrated for mosquito management.

Abstract:Mosquitocidal toxin 1 (Mtx1) was synthesized during vegetative phase of Bacillus sphaericus and it had been proved to have higher activity to Aedes spp. larvae and Binary toxin (Bin) resistance Culex larvae. The truncated 97kDa Mtx1 with a deletion of the signal peptide and the Cyt1Aa crystal protein, a 27.3kDa δ-endotoxin from Bacillus thuringiensis subsp. israelensis (Bti), were purified from Escherichia coli and B. thuringiensis recombinant strains respectively. Both purified toxins had high toxicity against Culex quinquefasciatus larvae. Bioassay result revealed the purified Mtx1 toxin had high toxicity against the target mosquito larvae, with LC₅₀ of 45.2ng/mL. However, the mixture of Mtx1 and Cyt1Aa exhibited higher toxicity against the mosquito larvae, with a lowest LC₅₀ value of 20.19ng/mL at the ratio of 1∶3. (Mtx1:Cyt1Aa). The calculated synergistic factor of different mixtures suggested a strong synergistic effect between Cyt1Aa toxin and Mtx1. Furthermore, the presence of Cyt1Aa in the mixture could induce early larval mortality, enhancing the activity of Mtx1 to the target mosquito larvae. The synergistic effect of Cyt1Aa on mortality of Mtx1 to mosquito larvae might be caused by the damage of the larval midgut-hemocoel barrier induced by the activated Cyt1Aa toxin, which enhanced the specific pathogenesis of Mtx1 on mosquito larvae. It is suggested that the co-application of Mtx1 and Cyt1Aa in future will be integrated for mosquito management.

Abstract:To study adaptive protein variation of H. pylori after colonization in Mongolian gerbils, Firstly, Clinical isolated strain M0 of H. pylori were inoculated into Mongolian gerbils and acclimated through serial passages in vivo for procuring an adaptive colonization H. pylori strain. Then, two-dimensional electrophoresis (2-DE) and mass spectrometry(MS) was taken to separate and identify the global proteins significantly changed between H. pylori strain M0and the adaptive strain. Through serial passages the infectious rate increased from about 2/10 to 9/10 and a adaptive colonization strain M₁₃ has been abtained. Comparative proteomic technology display that the proteinogram of H. pylori have changed after colonization in gerbils. Out of 5 differential protein spots cut out of gels for MALDI-TOF-MS identification, 4 spots were successfully identified, among which, Icd, RfaD and HP0318 were significantly higher in M₁₃ compared with M₀, while only HypB were found in M₁₃. So far, HP0318 is a conserved hypothetical protein. These proteins may be important factors of H. pylori for adaptive colonization.

Abstract:To study adaptive protein variation of H. pylori after colonization in Mongolian gerbils, Firstly, Clinical isolated strain M0 of H. pylori were inoculated into Mongolian gerbils and acclimated through serial passages in vivo for procuring an adaptive colonization H. pylori strain. Then, two-dimensional electrophoresis (2-DE) and mass spectrometry(MS) was taken to separate and identify the global proteins significantly changed between H. pylori strain M0and the adaptive strain. Through serial passages the infectious rate increased from about 2/10 to 9/10 and a adaptive colonization strain M₁₃ has been abtained. Comparative proteomic technology display that the proteinogram of H. pylori have changed after colonization in gerbils. Out of 5 differential protein spots cut out of gels for MALDI-TOF-MS identification, 4 spots were successfully identified, among which, Icd, RfaD and HP0318 were significantly higher in M₁₃ compared with M₀, while only HypB were found in M₁₃. So far, HP0318 is a conserved hypothetical protein. These proteins may be important factors of H. pylori for adaptive colonization.

Abstract:The fusion protein (F) and attachment glycoprotein (G) of Nipah virus (NiV) are important for the virus to infect cells and induce protective immunity. In this study, the NiV F1 and G gene fragments without the sequences of signal peptide and transmembrane domain were amplified by PCR, then cloned into E.coli expression vector pGEX-6P-1 and modified baculovirus vector, respectively. After induction by IPTG, NiV F1 and G proteins were efficiently expressed in E.coli when analyzed by SDS-PAGE, both showing good reactivity with the rabbit antiserum anti-NiV serum in Western blot. The expression of NiV F1 and G in baculovirus system were also detected by indirect immunofluorescent assay (IFA) of fixed Sf9 cells monolayer infected with the recombinant baculoviruses expressing F1 and G. Furthermore the anti-F1 and anti-G hyperimmune sera were prepared by immunization of rabbits respectively with purified E.coli-expressed F1 and G proteins. Western blot and IFA as well as ELISA showed that antisera against both protein had high titers with good reactivity and specificity. The present study has provided a base for development of diagnostic reagents for detection of NiV infection.

Abstract:The fusion protein (F) and attachment glycoprotein (G) of Nipah virus (NiV) are important for the virus to infect cells and induce protective immunity. In this study, the NiV F1 and G gene fragments without the sequences of signal peptide and transmembrane domain were amplified by PCR, then cloned into E.coli expression vector pGEX-6P-1 and modified baculovirus vector, respectively. After induction by IPTG, NiV F1 and G proteins were efficiently expressed in E.coli when analyzed by SDS-PAGE, both showing good reactivity with the rabbit antiserum anti-NiV serum in Western blot. The expression of NiV F1 and G in baculovirus system were also detected by indirect immunofluorescent assay (IFA) of fixed Sf9 cells monolayer infected with the recombinant baculoviruses expressing F1 and G. Furthermore the anti-F1 and anti-G hyperimmune sera were prepared by immunization of rabbits respectively with purified E.coli-expressed F1 and G proteins. Western blot and IFA as well as ELISA showed that antisera against both protein had high titers with good reactivity and specificity. The present study has provided a base for development of diagnostic reagents for detection of NiV infection.

Abstract:Pseudomonas putida DLL-1 is a high effective methyl parathion(MP) degrading strain, which is also of chemotaxis to MP. cheA, responsible for coding histidine kinase, plays an important role in bacterial chemotaxis signal transduction. In order to study the effect of bacterial chemotaxis on in-situ biodegradation of pesticides, cheA in chromosome of P. putida DLL-1 was mutated by gene-targeting and successfully obtained a MP-chemotaxis mutant DAK. The mutant DAK shows the same growth ability as wild-type DLL-1 in LB medium. The degrading rate of 50 mg/kg MP in non-sterile and sterile soil of chemotaxis mutant DAK is about 20%～30% lower than that of wild-type bacterial DLL-1. Lose of chemotaxis in DLL-1 would decline its degradation ability of MP. It was demonstrated that chemotaxis plays an important role in the in-situ biodegradation of pesticides.

Abstract:Pseudomonas putida DLL-1 is a high effective methyl parathion(MP) degrading strain, which is also of chemotaxis to MP. cheA, responsible for coding histidine kinase, plays an important role in bacterial chemotaxis signal transduction. In order to study the effect of bacterial chemotaxis on in-situ biodegradation of pesticides, cheA in chromosome of P. putida DLL-1 was mutated by gene-targeting and successfully obtained a MP-chemotaxis mutant DAK. The mutant DAK shows the same growth ability as wild-type DLL-1 in LB medium. The degrading rate of 50 mg/kg MP in non-sterile and sterile soil of chemotaxis mutant DAK is about 20%～30% lower than that of wild-type bacterial DLL-1. Lose of chemotaxis in DLL-1 would decline its degradation ability of MP. It was demonstrated that chemotaxis plays an important role in the in-situ biodegradation of pesticides.

Abstract:To investigate the immune effects of combined DNA immunization of human interleukin 12 and Mycobacterium tuberculosis ESAT-6 antigen. Thirty BALB/c mice were divided into 5 groups. They were immunized inter-muscularly with 100μL solution of normal saline (group A), pcDNA3.1 control plasmid (group B), pcESAT-6 plasmid (group D) and pcIL-12+pcESAT-6 plasmid (group E), respectively, for three times with 2 weeks interval. At the last time immunization, group C mice were immunized intra-dermally with 106CFU BCG suspended in normal saline in a volume of 100μL. After the last immunization 14 and 28 days, groups of mice (three mice per group) were sacrificed and separated their serum and splenocytes at once, respectively. The sera were cryopreserved for antigen-specific antibody production tests with ELISA. The separated mice spleen cells were cultured with completed RPMI 1640 medium in vitro. Specific splenocytes proliferation responses to TB-PPD antigen were measured by XTT colorimetry after five days culture. Another part of the culture spleen cells were stimulated with TB-PPD antigen for three days, then collected the supernatants to determine IFN-γ and IL-4 secretions level by ELISA test as well, respectively. The results show that both ESAT-6 immunization and IL-12+ESAT-6 combined immunization induce significantly anti-TB-PPD antibody production. Further, the antigen-specific splenocytes proliferation and IFN-γ production level of IL-12+ESAT-6 combined immunization are higher than pcESAT-6 or BCG immunization alone (P<0.05). However, it displays no distinct differences of IL-4 secretion among ESAT-6, BCG and IL-12+ESAT-6 combined immunization groups. It indicates that ESAT-6 immunization combined with IL-12 could induce much stronger specific cellular immunity than only ESAT-6 or conventional BCG immunization. (Therefore, codelivery of an IL-12 scereting plasmid with M. tuberculosis antigen may be a potent strategy for enhancing cellular immunity against M. tuberculosis.

Abstract:To investigate the immune effects of combined DNA immunization of human interleukin 12 and Mycobacterium tuberculosis ESAT-6 antigen. Thirty BALB/c mice were divided into 5 groups. They were immunized inter-muscularly with 100μL solution of normal saline (group A), pcDNA3.1 control plasmid (group B), pcESAT-6 plasmid (group D) and pcIL-12+pcESAT-6 plasmid (group E), respectively, for three times with 2 weeks interval. At the last time immunization, group C mice were immunized intra-dermally with 106CFU BCG suspended in normal saline in a volume of 100μL. After the last immunization 14 and 28 days, groups of mice (three mice per group) were sacrificed and separated their serum and splenocytes at once, respectively. The sera were cryopreserved for antigen-specific antibody production tests with ELISA. The separated mice spleen cells were cultured with completed RPMI 1640 medium in vitro. Specific splenocytes proliferation responses to TB-PPD antigen were measured by XTT colorimetry after five days culture. Another part of the culture spleen cells were stimulated with TB-PPD antigen for three days, then collected the supernatants to determine IFN-γ and IL-4 secretions level by ELISA test as well, respectively. The results show that both ESAT-6 immunization and IL-12+ESAT-6 combined immunization induce significantly anti-TB-PPD antibody production. Further, the antigen-specific splenocytes proliferation and IFN-γ production level of IL-12+ESAT-6 combined immunization are higher than pcESAT-6 or BCG immunization alone (P<0.05). However, it displays no distinct differences of IL-4 secretion among ESAT-6, BCG and IL-12+ESAT-6 combined immunization groups. It indicates that ESAT-6 immunization combined with IL-12 could induce much stronger specific cellular immunity than only ESAT-6 or conventional BCG immunization. (Therefore, codelivery of an IL-12 scereting plasmid with M. tuberculosis antigen may be a potent strategy for enhancing cellular immunity against M. tuberculosis.

Abstract:Three actinophages, ΦHAU7、ΦHAU9 and ΦHAU11, were isolated from soil using Micromonospora sp. 40027 as a indicator strain. Three phages showed very narrow host-ranges. Three phages could infect both Micromonospora sp. 40027 and Micromonospora A-M-01, ΦHAU9 and ΦHAU11 could also form plaques on Micromonospora purprea. All of the three phage particles have the hexagonal heads and tails; the plaque formation of three phages on Micromonospora sp. 40027 were best on DNA medium with addition of 32mM Ca²⁺ and 30mM Mg²⁺; ΦHAU7 was stable at pH 6～12, and other phages were stable at pH 6～10; the suitable incubation temperature for the propagation of three phages was between 28℃～37℃; 53% of ΦHAU7 remained viable and none of other phages was alive when they were incubated at 60℃ for 30min. Restriction digestion analysis of genomes of three phages indicated that they were all double-stranded DNA with cohesive ends, their genome size are ca. 60 kb、58 kb and 55kb, respectively.

Abstract:Three actinophages, ΦHAU7、ΦHAU9 and ΦHAU11, were isolated from soil using Micromonospora sp. 40027 as a indicator strain. Three phages showed very narrow host-ranges. Three phages could infect both Micromonospora sp. 40027 and Micromonospora A-M-01, ΦHAU9 and ΦHAU11 could also form plaques on Micromonospora purprea. All of the three phage particles have the hexagonal heads and tails; the plaque formation of three phages on Micromonospora sp. 40027 were best on DNA medium with addition of 32mM Ca²⁺ and 30mM Mg²⁺; ΦHAU7 was stable at pH 6～12, and other phages were stable at pH 6～10; the suitable incubation temperature for the propagation of three phages was between 28℃～37℃; 53% of ΦHAU7 remained viable and none of other phages was alive when they were incubated at 60℃ for 30min. Restriction digestion analysis of genomes of three phages indicated that they were all double-stranded DNA with cohesive ends, their genome size are ca. 60 kb、58 kb and 55kb, respectively.

Abstract:S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on Bt cell surface. Four recombinant plasmids were constructed by replacing 3′- terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene (npp). The four resulting plasmids were pSNP (harboring fusion gene ctc-np)，pCSA-SNP (harboring fusion gene csa-ctc-np，csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface)，pCTC-NPP (harboring fusion gene ctc-npp) and pCSNPP (harboring fusion gene csa-ctc-npp). Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid-free derivative strain BMB171. The resulting strains were BN (harboring pSNP)，BCN (harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon)，C-S (harboring pCSA-SNP)，BCCN (harboring pCTC-NPP and pMIL-CSA) and CN (harboring pCSNPP). The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains，respectively. After immunizing mice with vegetative cells of five recombinant strains respectively，five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). Meanwhile these assays showed recombinant strain CN (harboring fusion gene csa-ctc-npp) exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-* (* denotes heterologous antigen gene) which means the central part of S-layer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable，oral，veterinary vaccine with Bt S-layer protein CTC surface display system.

Abstract:S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on Bt cell surface. Four recombinant plasmids were constructed by replacing 3′- terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene (npp). The four resulting plasmids were pSNP (harboring fusion gene ctc-np)，pCSA-SNP (harboring fusion gene csa-ctc-np，csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface)，pCTC-NPP (harboring fusion gene ctc-npp) and pCSNPP (harboring fusion gene csa-ctc-npp). Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid-free derivative strain BMB171. The resulting strains were BN (harboring pSNP)，BCN (harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon)，C-S (harboring pCSA-SNP)，BCCN (harboring pCTC-NPP and pMIL-CSA) and CN (harboring pCSNPP). The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains，respectively. After immunizing mice with vegetative cells of five recombinant strains respectively，five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). Meanwhile these assays showed recombinant strain CN (harboring fusion gene csa-ctc-npp) exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-* (* denotes heterologous antigen gene) which means the central part of S-layer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable，oral，veterinary vaccine with Bt S-layer protein CTC surface display system.

Abstract:The influences of avian reovirus (ARV) infection at 1 day of age on bursa development，antibody responses after vaccinations to avian influenza virus (AIV)，Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV)，and pathogenecity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection in 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa，but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with ARV were still full protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were full protected from death after v-IBDV challenges，the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Supprisingly，ARV infection at ages of 1～7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chicknes with no ARV infection. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.

Abstract:The influences of avian reovirus (ARV) infection at 1 day of age on bursa development，antibody responses after vaccinations to avian influenza virus (AIV)，Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV)，and pathogenecity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection in 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa，but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with ARV were still full protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were full protected from death after v-IBDV challenges，the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Supprisingly，ARV infection at ages of 1～7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chicknes with no ARV infection. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.

Abstract:The epitope-G₁ gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G₁ recombinant plasmid successfully. The pGEX-G₁ was transformed into E.coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G₁ gene were obtained, which included reaction temperature 16℃, induction time 18h and IPTG concentration 0.1mmol/L. The soluble target protein was purified with Glutathione Sepharose TM^4B and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM^4B and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD₄₉₀ was 1.813±0.231, while the average of OD₄₉₀ from 12 negative sera was 0.359±0.032, and the distinction was very remarkable (P<0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD₄₉₀ detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324±0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.

Abstract:The epitope-G₁ gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G₁ recombinant plasmid successfully. The pGEX-G₁ was transformed into E.coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G₁ gene were obtained, which included reaction temperature 16℃, induction time 18h and IPTG concentration 0.1mmol/L. The soluble target protein was purified with Glutathione Sepharose TM^4B and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM^4B and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD₄₉₀ was 1.813±0.231, while the average of OD₄₉₀ from 12 negative sera was 0.359±0.032, and the distinction was very remarkable (P<0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD₄₉₀ detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324±0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.

Abstract:The full-length bovine interferon gamma(BoIFN-γ) cDNA，including the secretion signal peptide coding region was recloned into baculovirus honor vectors pFastBac^TM1 of Bac-To-Bac system. These recombinant plasmids，pFastBac^TM1-BoIFN-γ，were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids，rBacmid-BoIFN-γ. Recombinant baculovirus，rBac-BoIFN-γ，was generated for expressing BoIFN-γ，by transfecting recombinant Bacmid-BoIFN-γ with CellfectinRReagen into sf9 insect cells. BoIFN-γ efficiently expressed by recombinant baculovirus in sf9 cells was testified by indirect immunofluorescence assay and indirect ELISA with monoclonal antibody against Bovine interferon-γ. Furthermore，VSV*GFP，recombinant Vesicular Stomatitis Virus expressing green fluorescence protein and MDBK were used to determine the anti-viral activity of rBoIFN-γ. The result shows rBoIFN-γ could inhibit the replication of the VSV*GFP in MDBK cells and the antiviral activity of supernatant was 2×10⁵IU/mL. The antiviral activity of rBoIFN-γ could be blocked by anti-BoIFN-γ mouse serum. The results demonstrated that the recombinant baculovirus could express BoIFN-γ efficiently and rBoIFN-γ had high antiviral activity.

Abstract:The full-length bovine interferon gamma(BoIFN-γ) cDNA，including the secretion signal peptide coding region was recloned into baculovirus honor vectors pFastBac^TM1 of Bac-To-Bac system. These recombinant plasmids，pFastBac^TM1-BoIFN-γ，were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids，rBacmid-BoIFN-γ. Recombinant baculovirus，rBac-BoIFN-γ，was generated for expressing BoIFN-γ，by transfecting recombinant Bacmid-BoIFN-γ with CellfectinRReagen into sf9 insect cells. BoIFN-γ efficiently expressed by recombinant baculovirus in sf9 cells was testified by indirect immunofluorescence assay and indirect ELISA with monoclonal antibody against Bovine interferon-γ. Furthermore，VSV*GFP，recombinant Vesicular Stomatitis Virus expressing green fluorescence protein and MDBK were used to determine the anti-viral activity of rBoIFN-γ. The result shows rBoIFN-γ could inhibit the replication of the VSV*GFP in MDBK cells and the antiviral activity of supernatant was 2×10⁵IU/mL. The antiviral activity of rBoIFN-γ could be blocked by anti-BoIFN-γ mouse serum. The results demonstrated that the recombinant baculovirus could express BoIFN-γ efficiently and rBoIFN-γ had high antiviral activity.

Abstract:Vibrio parahaemolyticus is a gram-negative，halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin(TDH)，TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus，it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections，and its pathogenesis.

Abstract:Vibrio parahaemolyticus is a gram-negative，halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin(TDH)，TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus，it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections，and its pathogenesis.

Abstract:To express the recombinant protein MOMP_VD2-VD3 of Chlamydia pneumoniae，and research on the immunocompetence of the MOMPVD2-VD3 to support serodiagnosis，PCR and gene recombinant technique was used to clone the targeted DNA fragment from a strain AR-39. The recombinant plasmid was induced in E.coli BL21 after having constructed the prokaryotic expression system，then the immunocompetence of the expression product was analyzed by Western blot and indirected ELISA which is based on the animal experimentation. A group of control sera and 126 sera from patients with coronary heart disease were examined by using ELISAs based on the recombinant protein (MOMP_VD2-VD3)，and then the results were evaluated comparing with a commercial ELISAs kit. The results of the Western blot and indirected ELISA showed ompAVD2-VD3 gene inserted in pET30a could express a recombinant protein with the molecular weight of 24kDa in BL21 and specifically reacted with the antibodies against the MOMP. Specific humoral response was elicited after immune the BALB/c mouse with protein and the specific antibody titer was more than 1∶20480. Using a panel of control sera，the participation of the recombinant antigen，the sensitivity and the specificity of the indirected ELISAs were 100% respectively. Comparisons between two methods in detecting 126 sero samples，the concordance of two tests was 96.3%. The results reported here show that the recombinant protein with excellent immunocompetence could benefit the research on the serodiagnosis to Chlamydia pneumoniae.

Abstract:To express the recombinant protein MOMP_VD2-VD3 of Chlamydia pneumoniae，and research on the immunocompetence of the MOMPVD2-VD3 to support serodiagnosis，PCR and gene recombinant technique was used to clone the targeted DNA fragment from a strain AR-39. The recombinant plasmid was induced in E.coli BL21 after having constructed the prokaryotic expression system，then the immunocompetence of the expression product was analyzed by Western blot and indirected ELISA which is based on the animal experimentation. A group of control sera and 126 sera from patients with coronary heart disease were examined by using ELISAs based on the recombinant protein (MOMP_VD2-VD3)，and then the results were evaluated comparing with a commercial ELISAs kit. The results of the Western blot and indirected ELISA showed ompAVD2-VD3 gene inserted in pET30a could express a recombinant protein with the molecular weight of 24kDa in BL21 and specifically reacted with the antibodies against the MOMP. Specific humoral response was elicited after immune the BALB/c mouse with protein and the specific antibody titer was more than 1∶20480. Using a panel of control sera，the participation of the recombinant antigen，the sensitivity and the specificity of the indirected ELISAs were 100% respectively. Comparisons between two methods in detecting 126 sero samples，the concordance of two tests was 96.3%. The results reported here show that the recombinant protein with excellent immunocompetence could benefit the research on the serodiagnosis to Chlamydia pneumoniae.

Abstract:Altering -1 frameshifting efficiency of L-A virus in yeast T158c/S14a will result in loss of M1 virus and reduction of toxin K1，which induced a diminished of inhibition zone. According to the size of inhibition zone on methylene blue flat with low pH，a model for screening anti-viral agents was developed. The conditions of assaying killing activity by well test assay were explored. With assaying the killing activity in different pH and temperature，it was found that the optimum pH range of 4.3～4.7 and the optimum temperature range of 20～22℃ in this screening model. To utilize this screening model，Several Chinese crude drugs had been studied and were found Flos Lonicerae and Rhizoma Cimicifugae have anti-viral effect. Such a model was to establish foundation for high throughput screening of anti-viral agents.

Abstract:Altering -1 frameshifting efficiency of L-A virus in yeast T158c/S14a will result in loss of M1 virus and reduction of toxin K1，which induced a diminished of inhibition zone. According to the size of inhibition zone on methylene blue flat with low pH，a model for screening anti-viral agents was developed. The conditions of assaying killing activity by well test assay were explored. With assaying the killing activity in different pH and temperature，it was found that the optimum pH range of 4.3～4.7 and the optimum temperature range of 20～22℃ in this screening model. To utilize this screening model，Several Chinese crude drugs had been studied and were found Flos Lonicerae and Rhizoma Cimicifugae have anti-viral effect. Such a model was to establish foundation for high throughput screening of anti-viral agents.

Abstract:The fermentation rates of mannitol in toxigenic and non-toxigenic El Tor strains of Vibrio cholerae are obviously different，which is a valuable indicator in the rapid identification of toxigenic strain. To determine the regulating role of mtlR in transcription of mannitol PTS operon in V. cholerae，and whether it plays a role in the ferment difference of the toxigenic and non-toxigenic strains，the mtlR deletion mutants from the mannitol rapid-ferment strain (non-toxigenic strain) and slow-ferment strain (toxigenic strain) were constructed. Comparisons of the growth in M9 containing 0.2% mannitol as the sole carbon source and pH change in mannitol fermentation media of these wild strains and their mutants，indicated that mtlR is a repressor. Its repression in mtlCBA transcription was further verified with the analyses of quantitative reverse-transcriptional PCR. However，the regulation of mtlR is not the immediate cause of the ferment difference of the toxigenic and non-toxigenic strains. The study also provides the necessary data in the analyses of mannitol ferment difference between the toxigenic and non-toxigenic V. cholerae strains.

Abstract:The fermentation rates of mannitol in toxigenic and non-toxigenic El Tor strains of Vibrio cholerae are obviously different，which is a valuable indicator in the rapid identification of toxigenic strain. To determine the regulating role of mtlR in transcription of mannitol PTS operon in V. cholerae，and whether it plays a role in the ferment difference of the toxigenic and non-toxigenic strains，the mtlR deletion mutants from the mannitol rapid-ferment strain (non-toxigenic strain) and slow-ferment strain (toxigenic strain) were constructed. Comparisons of the growth in M9 containing 0.2% mannitol as the sole carbon source and pH change in mannitol fermentation media of these wild strains and their mutants，indicated that mtlR is a repressor. Its repression in mtlCBA transcription was further verified with the analyses of quantitative reverse-transcriptional PCR. However，the regulation of mtlR is not the immediate cause of the ferment difference of the toxigenic and non-toxigenic strains. The study also provides the necessary data in the analyses of mannitol ferment difference between the toxigenic and non-toxigenic V. cholerae strains.

Abstract:Metagenome DNA was extracted from Halichondria rugosa which was collected from South China Sea and kept in -4℃. PKS gene fragment was amplified using PCR with KS domain primers in PKS gene. A DNA fragment about 671bp in length was obtained by PCR. The PCR product was measured by agrose gel electrophoresis. Then the product was recovered from gel and cloned into pUCm-T vector. After that vectors were transformed into competent cells (DH5α). PKS gene fragment in positive clones was sequenced. Consequently，the corresponding amino acid sequence was deduced based on nucleotide sequence. BLAST analysis showed that the homology of this amino acid sequence with that deduced from KS domain of PKS gene in Rhodobacterales bacterium was up to 96%. Phylogenetic analysis indicated that the obtained PKS gene belongs to trans-AT KS domains. Meanwhile the result demonstrated the diversity and differences of microorganisms associated with and around sponge in different sea area. It is the first time to find bacterial PKS gene in sponge Halichondria rugosa，which provide powerful proof to the microbial origin hypothesis of sponge active compounds. At the same time，this study lay basis for the utilization of uncultured microorganisms associated with sponge from the aspect of genes.

Abstract:Metagenome DNA was extracted from Halichondria rugosa which was collected from South China Sea and kept in -4℃. PKS gene fragment was amplified using PCR with KS domain primers in PKS gene. A DNA fragment about 671bp in length was obtained by PCR. The PCR product was measured by agrose gel electrophoresis. Then the product was recovered from gel and cloned into pUCm-T vector. After that vectors were transformed into competent cells (DH5α). PKS gene fragment in positive clones was sequenced. Consequently，the corresponding amino acid sequence was deduced based on nucleotide sequence. BLAST analysis showed that the homology of this amino acid sequence with that deduced from KS domain of PKS gene in Rhodobacterales bacterium was up to 96%. Phylogenetic analysis indicated that the obtained PKS gene belongs to trans-AT KS domains. Meanwhile the result demonstrated the diversity and differences of microorganisms associated with and around sponge in different sea area. It is the first time to find bacterial PKS gene in sponge Halichondria rugosa，which provide powerful proof to the microbial origin hypothesis of sponge active compounds. At the same time，this study lay basis for the utilization of uncultured microorganisms associated with sponge from the aspect of genes.

Abstract:The chromosomal replication origins (oriC) have been investigated in Gram-positive eubacteria actinomycetes，including Streptomyces，Mycobacterium and Amycolatopsis，and reveal various DnaA-boxes and AT-rich sequences between the conserved dnaA and dnaN genes. Actinomadura yumaensis NRRL12515 is a producer of anthelmintic polyether maduramicin. In this paper，cloning，sequencing and functional studies of its oriC have been carried out. A pair of oligo-nucleotide primers，based on the conserved sequences of dnaA and dnaN，was used to PCR amplification. A～1.3kb DNA band was detected on agarose gel. Subsequently cloning in an E. coli plasmid pBluescriptⅡ SK(+) and sequencing showed 1265bp，which contained 919bp between dnaA and dnaN genes. 14 DnaA-boxes with conserved 9bp sequence (T/C)(T/C)GTCC(A/C)CA and two 13bp AT-rich regions (GAAAAATCCCAAG，AAGAAAAAACTCA)，were found on the sequence，indicating the oriC of A. yumaensis NRRL12515. Phylogenetic trees based on the sequences of oriC and of 16S rRNA genes of the four actinomycetes species show a similar pattern，suggesting that oriC sequences also reflected well the relationship between actinomycetes species. An E. coli plasmid pOR1，containing the oriC，actinomycetes selection markers tsr and melC，was introduced into Streptomyces coelicolor M145 by conjugal transfer. Transformants were obtained，and plasmids DNA were isolated and detected as low copy number，suggesting a functional mini-chromosome in Streptomyces.

Abstract:The chromosomal replication origins (oriC) have been investigated in Gram-positive eubacteria actinomycetes，including Streptomyces，Mycobacterium and Amycolatopsis，and reveal various DnaA-boxes and AT-rich sequences between the conserved dnaA and dnaN genes. Actinomadura yumaensis NRRL12515 is a producer of anthelmintic polyether maduramicin. In this paper，cloning，sequencing and functional studies of its oriC have been carried out. A pair of oligo-nucleotide primers，based on the conserved sequences of dnaA and dnaN，was used to PCR amplification. A～1.3kb DNA band was detected on agarose gel. Subsequently cloning in an E. coli plasmid pBluescriptⅡ SK(+) and sequencing showed 1265bp，which contained 919bp between dnaA and dnaN genes. 14 DnaA-boxes with conserved 9bp sequence (T/C)(T/C)GTCC(A/C)CA and two 13bp AT-rich regions (GAAAAATCCCAAG，AAGAAAAAACTCA)，were found on the sequence，indicating the oriC of A. yumaensis NRRL12515. Phylogenetic trees based on the sequences of oriC and of 16S rRNA genes of the four actinomycetes species show a similar pattern，suggesting that oriC sequences also reflected well the relationship between actinomycetes species. An E. coli plasmid pOR1，containing the oriC，actinomycetes selection markers tsr and melC，was introduced into Streptomyces coelicolor M145 by conjugal transfer. Transformants were obtained，and plasmids DNA were isolated and detected as low copy number，suggesting a functional mini-chromosome in Streptomyces.

Abstract:Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology，using Aspergillus oryzae AS3.951，Aspergillus flavus GIM3.18 and Aspergillus sojae AS3.495 as controls. First，genome DNA of the sixteen test strains were prepared by improved extraction method，and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band (approximately 20kb) in agarose gel electrophoresis，which conformed to the fact that these strains all belong to Aspergillus. OD₂₆₀/OD₂₈₀ of the prepared DNA ranged from 1.80 to 1.90，illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then，three appropriate primers (Primer1，Primer2，Primer5) for RAPD-PCR were screened from nine random primers，and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8～14 bands in their RADP-PCR patterns，where the number of the main bands was 4～9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns，where the percentage of polymorphic bands reached to 40.9% (74 bands). The similarity coefficient between the strains was calculated based on their RAPD-PCR patterns，ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primer1，Primer2 and Primer5. It basically corresponded to traditional morphological taxonomy，demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides，the aflatoxin-producing strains (GIM3.17，CICC2219，CICC2357，CICC2390，CICC2402，CICC2404) could be easily discriminated by RAPD molecular marker，whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently，RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.

Abstract:Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology，using Aspergillus oryzae AS3.951，Aspergillus flavus GIM3.18 and Aspergillus sojae AS3.495 as controls. First，genome DNA of the sixteen test strains were prepared by improved extraction method，and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band (approximately 20kb) in agarose gel electrophoresis，which conformed to the fact that these strains all belong to Aspergillus. OD₂₆₀/OD₂₈₀ of the prepared DNA ranged from 1.80 to 1.90，illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then，three appropriate primers (Primer1，Primer2，Primer5) for RAPD-PCR were screened from nine random primers，and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8～14 bands in their RADP-PCR patterns，where the number of the main bands was 4～9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns，where the percentage of polymorphic bands reached to 40.9% (74 bands). The similarity coefficient between the strains was calculated based on their RAPD-PCR patterns，ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primer1，Primer2 and Primer5. It basically corresponded to traditional morphological taxonomy，demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides，the aflatoxin-producing strains (GIM3.17，CICC2219，CICC2357，CICC2390，CICC2402，CICC2404) could be easily discriminated by RAPD molecular marker，whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently，RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.

Abstract:Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early，late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid，a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10⁵PFU and 2×10⁵PFU respectively. After 28 days，antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.

Abstract:Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early，late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid，a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10⁵PFU and 2×10⁵PFU respectively. After 28 days，antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.

Abstract:A steroid-converting fungus isolated from soil samples, was identified as Mucor racemosus according to its morphological characters. The application of M. racemosus for biotransformation of 4-ene-3-one steroids had been investigated to obtain hydroxylated derivatives of 4-ene-3-one steroids. The substrates were incubated with M. racemosus in rotary shaker (220 rpm) culture for a period of four days at 27℃. All of the fermentation media were exhaustively extracted with ethyl acetate and filtered to separate the both from the mycelium. The transformation products were separated on silica gel column chromatography. Each microbial metabolite was characterized by spectroscopic methods such as IR, MS and NMR. Fermentation of progesterone yielded 14α-hydroxypregn-4-en-3,20-dione and 7α,14α-dihydroxypregn-4-en-3,20-dione. Incubation of androstenedione resulted in three transformation products: 14,17-dihydroxyandrost-4-en-3-one, 14α,17β-dihydroxyandrost-4-en-3-one and 6α,17β-dihydroxyandrost-4-en-3-one. This fungus was found to biotransformation steroids. The results showed that the fermentation of 4-ene-3-one steroids with M. racemosus yielded mainly 14α-hydroxy steroids.

Abstract:A steroid-converting fungus isolated from soil samples, was identified as Mucor racemosus according to its morphological characters. The application of M. racemosus for biotransformation of 4-ene-3-one steroids had been investigated to obtain hydroxylated derivatives of 4-ene-3-one steroids. The substrates were incubated with M. racemosus in rotary shaker (220 rpm) culture for a period of four days at 27℃. All of the fermentation media were exhaustively extracted with ethyl acetate and filtered to separate the both from the mycelium. The transformation products were separated on silica gel column chromatography. Each microbial metabolite was characterized by spectroscopic methods such as IR, MS and NMR. Fermentation of progesterone yielded 14α-hydroxypregn-4-en-3,20-dione and 7α,14α-dihydroxypregn-4-en-3,20-dione. Incubation of androstenedione resulted in three transformation products: 14,17-dihydroxyandrost-4-en-3-one, 14α,17β-dihydroxyandrost-4-en-3-one and 6α,17β-dihydroxyandrost-4-en-3-one. This fungus was found to biotransformation steroids. The results showed that the fermentation of 4-ene-3-one steroids with M. racemosus yielded mainly 14α-hydroxy steroids.

Abstract:Atrazine(AT), a kind of herbicide for the pre and post-emergence control of annual and broad leaved weeds and perennial grasses, had been widely used in the world. However, the extensive use of atrazine had led to widespread environmental pollution. A bacterium strain SA1, which could degrade AT completely, was isolated from an atrazine-degrading consortium by long-time repeated alternative cultivation and plate striking. Combining cultural and physiobiochemical characteristics with 16S rDNA sequence analysis, SA1 was identified as Pseudomonas sp.. SA1 could use atrazine as the sole carbon, nitrogen and energy sources for growth, and the main product of AT biodegradation was cyanuric acid. AT degrading activity of SA1 was not affected by the addition of nitrogen resources. However, cyanuric acid could be degraded quickly to an undetectable level when glucose was added. The optimal temperature and pH value for SA1 growth was 37℃ and pH7, respectively. Atrazine could be degraded efficiently by the resting cells of SA1 under the conditions of 10℃～40℃ or pH value 4～11, and SA1 had a wide range of temperature and pH value for AT degradation when compared with ADP. atzABCD　and conserved sequence of tnpA gene of IS1071 could be amplified from SA1, and these genes could be lost during subculture.

Abstract:Atrazine(AT), a kind of herbicide for the pre and post-emergence control of annual and broad leaved weeds and perennial grasses, had been widely used in the world. However, the extensive use of atrazine had led to widespread environmental pollution. A bacterium strain SA1, which could degrade AT completely, was isolated from an atrazine-degrading consortium by long-time repeated alternative cultivation and plate striking. Combining cultural and physiobiochemical characteristics with 16S rDNA sequence analysis, SA1 was identified as Pseudomonas sp.. SA1 could use atrazine as the sole carbon, nitrogen and energy sources for growth, and the main product of AT biodegradation was cyanuric acid. AT degrading activity of SA1 was not affected by the addition of nitrogen resources. However, cyanuric acid could be degraded quickly to an undetectable level when glucose was added. The optimal temperature and pH value for SA1 growth was 37℃ and pH7, respectively. Atrazine could be degraded efficiently by the resting cells of SA1 under the conditions of 10℃～40℃ or pH value 4～11, and SA1 had a wide range of temperature and pH value for AT degradation when compared with ADP. atzABCD　and conserved sequence of tnpA gene of IS1071 could be amplified from SA1, and these genes could be lost during subculture.

Abstract:Preliminary statistics showed that there are more than one million species of microbes in marine environments that formed a dynamic genetic reservoir, among which the majority are not revealed and categorized due to barrier in cultivation techniques. However, the situation has changed in recent years because of the rapid development of phylogenetic studies based on small ribosomal RNA and rDNA sequencing independent to standard laboratory cultivation. These changes have significantly altered our understanding about microbial diversity and microbial ecology. In this review, we highlight some of recent progress and innovation in research on microbial diversity, and propose a metagenomic scheme as an alternative to overcome some of the barriers that still remain for exploitation of marine microbial diversity for its enormous potential in pharmaceutical applications. We believe that rapid progress in marine metagenomics allows direct access to the genomes of numerous non-cultivable microorganisms for their associated chemical prosperity.

Abstract:Preliminary statistics showed that there are more than one million species of microbes in marine environments that formed a dynamic genetic reservoir, among which the majority are not revealed and categorized due to barrier in cultivation techniques. However, the situation has changed in recent years because of the rapid development of phylogenetic studies based on small ribosomal RNA and rDNA sequencing independent to standard laboratory cultivation. These changes have significantly altered our understanding about microbial diversity and microbial ecology. In this review, we highlight some of recent progress and innovation in research on microbial diversity, and propose a metagenomic scheme as an alternative to overcome some of the barriers that still remain for exploitation of marine microbial diversity for its enormous potential in pharmaceutical applications. We believe that rapid progress in marine metagenomics allows direct access to the genomes of numerous non-cultivable microorganisms for their associated chemical prosperity.

Abstract:Selenium(Se) is a naturally occurring trace element. It is essential for humans and animals but is very toxic at higher concentrations and will make the selenium pollution. In four inorganic states of selenate［SeO₄^2- (VI)］, selenite［SeO₃^2- (IV)］, elemental selenium［Se° (0)］ and selenide［Se^2- (-II)］, selenium oxyanions including selenite and selenate are well known to be more bioavailable but higher toxic than other forms. Many bacteria have the capacity to reduce selenium oxyanions to red elemental ground granule, called red elemental selenium, which is likely to be Se-protein compound. It has lower toxicity comparing with selenite, but when the size is at 5～200 nm, its bioavailability to selenite was similar in terms of inducing seleno-enzymes in cultured cells and in Se-deficient rats. This provides the potential to gain the best manner to supply selenium for organisms and solve the pollution of selenium. So far, series of researches have been made to study this bacterial reduced process. Many substances such as carbon source, oxygen, elemental sulfur, glutathione, some oxidoreductases and transfer protein in membrane were found to affect or participate this biological dissimilatory reduction. This review presents the discovery of the biomorphic red elemental selenium, factors affecting the bacterial reduction of the selenium oxyanions, and the possible pathways to form red elemental selenium in bacteria.

Abstract:Selenium(Se) is a naturally occurring trace element. It is essential for humans and animals but is very toxic at higher concentrations and will make the selenium pollution. In four inorganic states of selenate［SeO₄^2- (VI)］, selenite［SeO₃^2- (IV)］, elemental selenium［Se° (0)］ and selenide［Se^2- (-II)］, selenium oxyanions including selenite and selenate are well known to be more bioavailable but higher toxic than other forms. Many bacteria have the capacity to reduce selenium oxyanions to red elemental ground granule, called red elemental selenium, which is likely to be Se-protein compound. It has lower toxicity comparing with selenite, but when the size is at 5～200 nm, its bioavailability to selenite was similar in terms of inducing seleno-enzymes in cultured cells and in Se-deficient rats. This provides the potential to gain the best manner to supply selenium for organisms and solve the pollution of selenium. So far, series of researches have been made to study this bacterial reduced process. Many substances such as carbon source, oxygen, elemental sulfur, glutathione, some oxidoreductases and transfer protein in membrane were found to affect or participate this biological dissimilatory reduction. This review presents the discovery of the biomorphic red elemental selenium, factors affecting the bacterial reduction of the selenium oxyanions, and the possible pathways to form red elemental selenium in bacteria.

Abstract:Contrast to planktonic cells, biofilms are complex communities of microorganisms that develop on biotic or abiotic surfaces. Most of bacteria can form biofilms under proper conditions. Once biofilm form, the inner bacteria cells often exhibit reduced antibiotic susceptibility than their free-floating counterparts, so conventional methods of killing bacteria, such as antibiotics and disinfections are often ineffective with them. Biofilms may cause huge economic loss in equipment damage, product contamination, energy losses and medical infections. Therefore, bacterial biofilm is evolving as a focal problem and an active area of research. As a relatively new area, the progress of biofilm science depends on the development of a satisfactory set of methods. But the classic methods to study planktonic bacteria cannot fulfill the biofilm research, one for which there are few widely accepted methodological standards. Even though biofilms are complicated physical-chemical-biological systems, experience demonstrates that accessible research methods are feasible. In this paper, the theories, principles, merits and limitations of some methods currently used in bacterial biofilm researches, involving artificial biofilm forming and biofilm measurement, were discussed.

Abstract:Contrast to planktonic cells, biofilms are complex communities of microorganisms that develop on biotic or abiotic surfaces. Most of bacteria can form biofilms under proper conditions. Once biofilm form, the inner bacteria cells often exhibit reduced antibiotic susceptibility than their free-floating counterparts, so conventional methods of killing bacteria, such as antibiotics and disinfections are often ineffective with them. Biofilms may cause huge economic loss in equipment damage, product contamination, energy losses and medical infections. Therefore, bacterial biofilm is evolving as a focal problem and an active area of research. As a relatively new area, the progress of biofilm science depends on the development of a satisfactory set of methods. But the classic methods to study planktonic bacteria cannot fulfill the biofilm research, one for which there are few widely accepted methodological standards. Even though biofilms are complicated physical-chemical-biological systems, experience demonstrates that accessible research methods are feasible. In this paper, the theories, principles, merits and limitations of some methods currently used in bacterial biofilm researches, involving artificial biofilm forming and biofilm measurement, were discussed.