Abstract:Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin- and ampicillin-resistance-encoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum.This vector carried the promoterless chloramphenicol acetyltranstersae(CAT) gene as a reporter downstream from useful cloning site. For testing this promoter-probe vector, C. glutamicum genomic DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique BglⅡ. Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed.After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.

Abstract:Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin- and ampicillin-resistance-encoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum.This vector carried the promoterless chloramphenicol acetyltranstersae(CAT) gene as a reporter downstream from useful cloning site. For testing this promoter-probe vector, C. glutamicum genomic DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique BglⅡ. Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed.After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.

Abstract:Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain isolated from an outbreak in the goose, seven pairs of primers were designed to amplify cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of NDV ZJI strain. The pNDV/ZJI with three helper plasmids, pCI-NP, pCI-P and pCI-L, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, infectious NDV ZJI strain was successfully rescued. The recombinant plasmid pNDV/ZJIFM was generated by converting the multi-basic amino acid sequence of the F0 protein cleavage region in pNDV/ZJI to the non-basic amino acid sequence characteristic of avirulent NDV strain. After cotransfection of the resultant plasmid and the three helper plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIFM, was generated. The mean death time (MDT) of NDV/ZJIFM was more than 120h and the intrancerebral pathogenicity index (ICPI) was 0.16, indicating that the rescued virus was highly attenuated. This attenuated genotype VIId NDV of goose origin could be a desirable vaccine in controlling the current epidemic of ND.

Abstract:Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain isolated from an outbreak in the goose, seven pairs of primers were designed to amplify cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of NDV ZJI strain. The pNDV/ZJI with three helper plasmids, pCI-NP, pCI-P and pCI-L, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, infectious NDV ZJI strain was successfully rescued. The recombinant plasmid pNDV/ZJIFM was generated by converting the multi-basic amino acid sequence of the F0 protein cleavage region in pNDV/ZJI to the non-basic amino acid sequence characteristic of avirulent NDV strain. After cotransfection of the resultant plasmid and the three helper plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIFM, was generated. The mean death time (MDT) of NDV/ZJIFM was more than 120h and the intrancerebral pathogenicity index (ICPI) was 0.16, indicating that the rescued virus was highly attenuated. This attenuated genotype VIId NDV of goose origin could be a desirable vaccine in controlling the current epidemic of ND.

Abstract:In order to explore the resistance and the staphylococcal chromosome cassette mec (SCCmec) types of Methicillin-resistant S. aureus (MRSA) in the area of Haikou, 686 strains of MRSA had been distinguished from 1174 strains of S. aureus using PBP2a testing. The resistance to the seven deputies of seven kinds antibiotics which in common use in clinic, including Oxacillin, Vancomycin，Doxycyclin, Amikacin, Erythromycin, Chloramphenicol，Ciprofloxacin, and SCCmec type of 58 strains had been tested using the K-B Agar diffuse，E-test and multiplex PCR strategy, and seven kinds of new SCCmec types were found in 17 strains. Their specialties of structure are: type-new3 possess four loci of A,F,H,M; New4 possess three loci of F,H,M; New5 possess three loci of D,B,M; New6 possess three loci of A,B,M; New7 possess four loci of H,E,C,M; New8 possess two loci of A,M; New9 possess three loci of A,C,M. All of them are different from the types reported. The strains carrying new SCCmec types are different from that carrying old SCCmec types in the epidemical distribution and resistance to the antibiotics: they were mostly isolated from the out-patients and have high level and wider range of resistance to antibiotics and deserve to pay more attention.

Abstract:In order to explore the resistance and the staphylococcal chromosome cassette mec (SCCmec) types of Methicillin-resistant S. aureus (MRSA) in the area of Haikou, 686 strains of MRSA had been distinguished from 1174 strains of S. aureus using PBP2a testing. The resistance to the seven deputies of seven kinds antibiotics which in common use in clinic, including Oxacillin, Vancomycin，Doxycyclin, Amikacin, Erythromycin, Chloramphenicol，Ciprofloxacin, and SCCmec type of 58 strains had been tested using the K-B Agar diffuse，E-test and multiplex PCR strategy, and seven kinds of new SCCmec types were found in 17 strains. Their specialties of structure are: type-new3 possess four loci of A,F,H,M; New4 possess three loci of F,H,M; New5 possess three loci of D,B,M; New6 possess three loci of A,B,M; New7 possess four loci of H,E,C,M; New8 possess two loci of A,M; New9 possess three loci of A,C,M. All of them are different from the types reported. The strains carrying new SCCmec types are different from that carrying old SCCmec types in the epidemical distribution and resistance to the antibiotics: they were mostly isolated from the out-patients and have high level and wider range of resistance to antibiotics and deserve to pay more attention.

Abstract:A gacA homologue, designated gacA_Xooc, was cloned from Xanthomonas oryzae pv. oryzicola (Xooc), a bacterium that causes leaf streak of rice, with degenerated primers by polymerase amplification reaction (PCR). NCBI blast search indicated that GacA_Xooc had a similar structure to that of other GacA proteins, and had a CheB（Chemotaxis response regulator containing a CheY-like receiver domain）domain. Sequence comparison showed that the gacA_Xooc was conserved in the Xanthomonas genus. Homology search revealed that the gacA_Xooc was 99.7% similarities to gacA（AY870457, this lab）of Xanthomonas oryzae pv. oryzae (Xoo). A gacA_Xooc disruption mutant was successfully generated by a single cross-over event, and confirmed by PCR and Southern blot. But the mutant still had strong pathogenicity，and its virulence was not obviously different from that of wild type strain. The gacA did not globally regulate metabolism in Xooc, which was different from DC3000 of P. syringae pv. tomato, CHA0 of P. fluorescens and IC1270 of Serratia plymuthica. Chemotaxis to 0.1% tryptone of the mutants was reduced compared to wild type strain. The results suggest that gacA_Xooc is involved chemotaxis of Xooc. Nevertheless, how gacA to regulate chemotaxis of Xooc, transcription and expression of genes involved in regulation still need to be further studied.

Abstract:A gacA homologue, designated gacA_Xooc, was cloned from Xanthomonas oryzae pv. oryzicola (Xooc), a bacterium that causes leaf streak of rice, with degenerated primers by polymerase amplification reaction (PCR). NCBI blast search indicated that GacA_Xooc had a similar structure to that of other GacA proteins, and had a CheB（Chemotaxis response regulator containing a CheY-like receiver domain）domain. Sequence comparison showed that the gacA_Xooc was conserved in the Xanthomonas genus. Homology search revealed that the gacA_Xooc was 99.7% similarities to gacA（AY870457, this lab）of Xanthomonas oryzae pv. oryzae (Xoo). A gacA_Xooc disruption mutant was successfully generated by a single cross-over event, and confirmed by PCR and Southern blot. But the mutant still had strong pathogenicity，and its virulence was not obviously different from that of wild type strain. The gacA did not globally regulate metabolism in Xooc, which was different from DC3000 of P. syringae pv. tomato, CHA0 of P. fluorescens and IC1270 of Serratia plymuthica. Chemotaxis to 0.1% tryptone of the mutants was reduced compared to wild type strain. The results suggest that gacA_Xooc is involved chemotaxis of Xooc. Nevertheless, how gacA to regulate chemotaxis of Xooc, transcription and expression of genes involved in regulation still need to be further studied.

Abstract:The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with HindⅢ, DNA fragments ranging from 50-100kb was collected and ligated to pCC1BAC vector. The ligation mixture was transformed into E.coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5kb, mostly ranging from 50-70kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase,Cmcellulase had been screened from the BAC library.The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity.

Abstract:The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with HindⅢ, DNA fragments ranging from 50-100kb was collected and ligated to pCC1BAC vector. The ligation mixture was transformed into E.coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5kb, mostly ranging from 50-70kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase,Cmcellulase had been screened from the BAC library.The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity.

Abstract:Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al., 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing Cyt1Aa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and Cyt1Aa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid.

Abstract:Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al., 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing Cyt1Aa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and Cyt1Aa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid.

Abstract:A total of 117 Salmonella strains isolated from retail meats and human in some regions of Jiangsu were assayed for their antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and the ability of horizontal transfer of the characterized antimicrobial resistance determinants via conjugation. 111 (94.9%) were resistant to at least two antimicrobial agents. Resistance against streptomycin 92.3% （108/117） was the most common. Whereas 59 (50.4%) were resistant to at least five antimicrobials. Twelve isolates showed resistance to six antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and kanamycin (R type ACSSuTK). A total of 15 different antimicrobial resistance genes were identified in 36 multidrug_resistant Salmonella isolates. While 30 (83%) of these isolates tested carried integrons ranging in size from 0.3 to 1.6 kb. The most common integron was 1.6 kb which carried aadA5 and dfr17 gene cassettes. Conjugation studies demonstrated that there was plasmid_mediated transfer of antimicrobial resistance genes. These data showed that the Salmonella isolates recovered from meats and human in some regions of Jiangsu were commonly resistant to multiple antimicrobials, and genes conferring antimicrobial resistance in Salmonella were often carried on integrons and plasmids and could be transmitted through conjugation. The mobile DNA elements might played very important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.

Abstract:A total of 117 Salmonella strains isolated from retail meats and human in some regions of Jiangsu were assayed for their antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and the ability of horizontal transfer of the characterized antimicrobial resistance determinants via conjugation. 111 (94.9%) were resistant to at least two antimicrobial agents. Resistance against streptomycin 92.3% （108/117） was the most common. Whereas 59 (50.4%) were resistant to at least five antimicrobials. Twelve isolates showed resistance to six antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and kanamycin (R type ACSSuTK). A total of 15 different antimicrobial resistance genes were identified in 36 multidrug_resistant Salmonella isolates. While 30 (83%) of these isolates tested carried integrons ranging in size from 0.3 to 1.6 kb. The most common integron was 1.6 kb which carried aadA5 and dfr17 gene cassettes. Conjugation studies demonstrated that there was plasmid_mediated transfer of antimicrobial resistance genes. These data showed that the Salmonella isolates recovered from meats and human in some regions of Jiangsu were commonly resistant to multiple antimicrobials, and genes conferring antimicrobial resistance in Salmonella were often carried on integrons and plasmids and could be transmitted through conjugation. The mobile DNA elements might played very important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.

Abstract:Antibacterial and cytotoxic activities were screened for marine bacteria which have been isolated from organism, sediments and seawater in China coastal area. The results showed that 42 isolates had antimicrobial activity and 12 isolates had cytotoxicity. Molecular phylogenetic analysis of marine bacteria with cytotoxicity based on 16S rRNA sequences indicated that they were belong to the genera Aerococcus, Agrobacterium, Alteromona, Bacillus, Exiguobacterium, Paracoccus,Pseudoalteromons, Rheinheimera. Furthermore, marine bacteria with cytotoxicity were also screened for PKS Ⅰ and NRPS genes which could be responsible for bioactive secondary metabolites biosynthesis, 4 strains having KS domain or A domain were obtained, which provide strong evidence that marine bioactive bacteria can produce natural products through PKS and NRPS pathways.

Abstract:Antibacterial and cytotoxic activities were screened for marine bacteria which have been isolated from organism, sediments and seawater in China coastal area. The results showed that 42 isolates had antimicrobial activity and 12 isolates had cytotoxicity. Molecular phylogenetic analysis of marine bacteria with cytotoxicity based on 16S rRNA sequences indicated that they were belong to the genera Aerococcus, Agrobacterium, Alteromona, Bacillus, Exiguobacterium, Paracoccus,Pseudoalteromons, Rheinheimera. Furthermore, marine bacteria with cytotoxicity were also screened for PKS Ⅰ and NRPS genes which could be responsible for bioactive secondary metabolites biosynthesis, 4 strains having KS domain or A domain were obtained, which provide strong evidence that marine bioactive bacteria can produce natural products through PKS and NRPS pathways.

Abstract:A Quantitative detection assay of acrA-mRNA of Escherichia coli ATCC25922 was developed by Quantitative Competitive RT-PCR. Target Standard(TS) which was same as target-templete acrA was amplified by PCR with P1 and P2 as primers. Internal Standard (IS) which was shorter 68bp then target-templete acrA was amplified by temperature-gradient-PCR with P1 and P3P2 as primers, whose annealing temperatures ranged from 55～65℃, and the most suitable annealing temperature was acquired at 56℃. Both TS and IS were largely amplified by PCR as above and extracted to store. Co-amplification with both TS and IS as templetes was optimized by temperature-gradient-PCR with P1 and P2 as primers, whose annealing temperatures were ranged from 55～65℃, and then the most suitable annealing temperature was also acquired at 56℃. Then co-amplification optimized as above was did again but with both cDNA of Escherichia coli (with target-templete acrA-cDNA copies unknown) and IS(10-fold serial dilution, and with IS copies known) as templates. The electrophoresis bands were photographed and analysed with UVIband and each band area was acquired, then linear regression analysis was did with SPSS11.5 and CurveExpert1.3 and a competitive curve was drawn as y=－0.345+0.097x. Results revealed that the two kinds of product electrophoresis bands of co-amplification, whose templates were both 10-fold diluted IS and cDNA, could be distinguished clearly in 1.5% agarose gel because of 68bp discrepancy, and showed lighteness dimming gradually with IS copies 10-fold diluting. With the competitive curve, the copies of acrA-mRNA in sample could be counted accurately and easily.

Abstract:A Quantitative detection assay of acrA-mRNA of Escherichia coli ATCC25922 was developed by Quantitative Competitive RT-PCR. Target Standard(TS) which was same as target-templete acrA was amplified by PCR with P1 and P2 as primers. Internal Standard (IS) which was shorter 68bp then target-templete acrA was amplified by temperature-gradient-PCR with P1 and P3P2 as primers, whose annealing temperatures ranged from 55～65℃, and the most suitable annealing temperature was acquired at 56℃. Both TS and IS were largely amplified by PCR as above and extracted to store. Co-amplification with both TS and IS as templetes was optimized by temperature-gradient-PCR with P1 and P2 as primers, whose annealing temperatures were ranged from 55～65℃, and then the most suitable annealing temperature was also acquired at 56℃. Then co-amplification optimized as above was did again but with both cDNA of Escherichia coli (with target-templete acrA-cDNA copies unknown) and IS(10-fold serial dilution, and with IS copies known) as templates. The electrophoresis bands were photographed and analysed with UVIband and each band area was acquired, then linear regression analysis was did with SPSS11.5 and CurveExpert1.3 and a competitive curve was drawn as y=－0.345+0.097x. Results revealed that the two kinds of product electrophoresis bands of co-amplification, whose templates were both 10-fold diluted IS and cDNA, could be distinguished clearly in 1.5% agarose gel because of 68bp discrepancy, and showed lighteness dimming gradually with IS copies 10-fold diluting. With the competitive curve, the copies of acrA-mRNA in sample could be counted accurately and easily.

Abstract:B.ovis 019 strain was identified by traditional methods and HOOF-Prints technique. Software DNAMAN was used to analyze phylogenetic tree for B.ovis 019 and reference strains of B. abortus 2308, B. melitensis 16M, B. suis 1330 and B. ovis 63/290. The results showed that the similarity between B.ovis 019 and B. ovis 63/290 was higher than that between B.ovis 019 and the other reference strains, which was in accordance with the results by conventional bacteriological methods. HOOF-Prints technique would be a promising method for identifying Brucella species and even biovars.

Abstract:B.ovis 019 strain was identified by traditional methods and HOOF-Prints technique. Software DNAMAN was used to analyze phylogenetic tree for B.ovis 019 and reference strains of B. abortus 2308, B. melitensis 16M, B. suis 1330 and B. ovis 63/290. The results showed that the similarity between B.ovis 019 and B. ovis 63/290 was higher than that between B.ovis 019 and the other reference strains, which was in accordance with the results by conventional bacteriological methods. HOOF-Prints technique would be a promising method for identifying Brucella species and even biovars.

Abstract:Lactobacillus plantarum ZS2058, which was screened from the Chinese traditional fermented vegetable，has the capacity to convert the linoleic acid(LA) into conjugated linoleic acid (CLA). Some specific isomers of CLA with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from free linoleic acid by washed cells of Lactobacillus plantarum ZS2058 under aerobic conditions. The produced CLA isomers are identified as the mixture of cis-9,trans-11-octadecadienoic acid (CLA1) trans-10, cis-12- octadecadienoic acid (CLA2),96.4% of which is CLA1. The washed cells of Lactobacillus plantarum ZS2058 producing high levels of c9,t11-CLA were obtained by cultivated in MRS media containing 0.5mg/mL linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After a 24-hour bioconversion at 37℃ with shaking (120 r/min), 312.4μg/mL c9,t11-CLA is produced. And after a 36-hour bioconversion, the content of c9,t11-CLA decreases while hydroxy-octadecaenoic acid increases. In addition, the c9,t11-CLA isomer can be transformed to hydroxy- octadecaenoic acid when the mixed CLA (c9,t11-CLA and t10,c12-CLA ) were used as the substrate ，which suggests that c9,t11-CLA is one of the intermediates of the bioconversion products from free LA by washed cells of Lactobacillus plantarum ZS2058.

Abstract:Lactobacillus plantarum ZS2058, which was screened from the Chinese traditional fermented vegetable，has the capacity to convert the linoleic acid(LA) into conjugated linoleic acid (CLA). Some specific isomers of CLA with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from free linoleic acid by washed cells of Lactobacillus plantarum ZS2058 under aerobic conditions. The produced CLA isomers are identified as the mixture of cis-9,trans-11-octadecadienoic acid (CLA1) trans-10, cis-12- octadecadienoic acid (CLA2),96.4% of which is CLA1. The washed cells of Lactobacillus plantarum ZS2058 producing high levels of c9,t11-CLA were obtained by cultivated in MRS media containing 0.5mg/mL linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After a 24-hour bioconversion at 37℃ with shaking (120 r/min), 312.4μg/mL c9,t11-CLA is produced. And after a 36-hour bioconversion, the content of c9,t11-CLA decreases while hydroxy-octadecaenoic acid increases. In addition, the c9,t11-CLA isomer can be transformed to hydroxy- octadecaenoic acid when the mixed CLA (c9,t11-CLA and t10,c12-CLA ) were used as the substrate ，which suggests that c9,t11-CLA is one of the intermediates of the bioconversion products from free LA by washed cells of Lactobacillus plantarum ZS2058.

Abstract:Brevibacterium flavum is used for the production of a number of amino acids in the biotechnology industry. The yield of producing a metabolite is ultimately limited by the ability of the central metabolism and the desired biosynthesis pathway. Pathway analysis is a very useful tool for metabolic engineering, which can be applied to increase the yield of a metabolite or channeling a metabolite into desired pathways. It does not require any kinetic parameters and only uses the Stoichiometric equations. Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303 at steady state was conducted in this paper. Theoretical yield and flux distribution for optimal pathway were determined.It is also concluded that pyruvate and acetyl-coenzyme A are the key nodes of the L-leucine biosynthesis pathway by analyzing the flux distributions of different modes. According to the pathway analysis, the production of L-leucine is expected to be raised by strengthening the flux of the key nodes (pyruvate and acetyl-coenzyme A) through changing the environmental factors. Because the flux of TCA cycle in Brevibacterium flavum TK0303 is weak, the production of L-leucine must be provided enough amido by adding glutamic acid to the fermentation medium. NH₄Ac is both a carbon source and a nitrogen source, which could be helpful to the production of L-leucine. The effects of glutamic acid and NH₄Ac on the production of L-leucine were further studied. The production of L-leucine increased 56% by adding glutamic acid. By improving the concentration of NH4Ac, the biosynthesis of L-leucine was greatly strengthened too. The results indicate that the flux of L-leucine can be largely increased by changing the chemical regulatory factors such as NH₄Ac and glutamic acid and the modes established by pathway analysis prove to be efficient to describe the metabolic network of L-leucine production by Brevibacterium flavum TK0303.

Abstract:Brevibacterium flavum is used for the production of a number of amino acids in the biotechnology industry. The yield of producing a metabolite is ultimately limited by the ability of the central metabolism and the desired biosynthesis pathway. Pathway analysis is a very useful tool for metabolic engineering, which can be applied to increase the yield of a metabolite or channeling a metabolite into desired pathways. It does not require any kinetic parameters and only uses the Stoichiometric equations. Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303 at steady state was conducted in this paper. Theoretical yield and flux distribution for optimal pathway were determined.It is also concluded that pyruvate and acetyl-coenzyme A are the key nodes of the L-leucine biosynthesis pathway by analyzing the flux distributions of different modes. According to the pathway analysis, the production of L-leucine is expected to be raised by strengthening the flux of the key nodes (pyruvate and acetyl-coenzyme A) through changing the environmental factors. Because the flux of TCA cycle in Brevibacterium flavum TK0303 is weak, the production of L-leucine must be provided enough amido by adding glutamic acid to the fermentation medium. NH₄Ac is both a carbon source and a nitrogen source, which could be helpful to the production of L-leucine. The effects of glutamic acid and NH₄Ac on the production of L-leucine were further studied. The production of L-leucine increased 56% by adding glutamic acid. By improving the concentration of NH4Ac, the biosynthesis of L-leucine was greatly strengthened too. The results indicate that the flux of L-leucine can be largely increased by changing the chemical regulatory factors such as NH₄Ac and glutamic acid and the modes established by pathway analysis prove to be efficient to describe the metabolic network of L-leucine production by Brevibacterium flavum TK0303.

Abstract:Gram-negative bacteria, global regulator QscR controls the expression of many virulence determinants, secondary metabolites, stationary phase genes and genes involved in biofilm formation through quorum sensing (QS) systems. QscR binds the promoter region of target genes and regulates the gene expression at the transcriptional level. Using homologous recombination technique a chromosomal qscR inactivated mutant strain M-18Q was constructed in Pseudomonas sp. M-18, a strain of plant-growth-promoting rhizobacteria, which could inhibit several soilborn phytopathogens by producing secondary metabolites including phenazine-1-carboxylic acid（PCA）and pyoluteorin (Plt) in one single strain. To further study the effect of QscR on the synthesis of Plt and PCA in the wild type strain M-18, the dynamic curves of Plt and PCA produced respectively by M-18 and M-18Q strains were measured in both KMB and PPM mediums .The synthesis of PCA was much more activated in the mutant than in the wild type both in KMB and PPM mediums. The PCA production in the mutant strain is four-to-six fold over that in the wild type in the PPM medium, reaching 480μg/mL, and three-to-five fold in the KMB medium, reaching 140μg/mL. The synthesis of Plt, however, was not detected in PPM medium and was nearly not influenced by the QscR protein in KMB medium. PCA production was inhibited but Plt biosynthesis was not altered after complementation with qscR gene in trans in the strain of M-18Q. The regulation of qscR gene on PCA production was further confirmed by the analysis of β-galactosidase activities from the translational phzA‘-'lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway. These results indicate that QscR can control PCA production negatively but not Plt production in M-18，and show that QscR functions as a global regulator to differently regulate the synthesis of PCA and Plt on the gene expression level.

Abstract:Gram-negative bacteria, global regulator QscR controls the expression of many virulence determinants, secondary metabolites, stationary phase genes and genes involved in biofilm formation through quorum sensing (QS) systems. QscR binds the promoter region of target genes and regulates the gene expression at the transcriptional level. Using homologous recombination technique a chromosomal qscR inactivated mutant strain M-18Q was constructed in Pseudomonas sp. M-18, a strain of plant-growth-promoting rhizobacteria, which could inhibit several soilborn phytopathogens by producing secondary metabolites including phenazine-1-carboxylic acid（PCA）and pyoluteorin (Plt) in one single strain. To further study the effect of QscR on the synthesis of Plt and PCA in the wild type strain M-18, the dynamic curves of Plt and PCA produced respectively by M-18 and M-18Q strains were measured in both KMB and PPM mediums .The synthesis of PCA was much more activated in the mutant than in the wild type both in KMB and PPM mediums. The PCA production in the mutant strain is four-to-six fold over that in the wild type in the PPM medium, reaching 480μg/mL, and three-to-five fold in the KMB medium, reaching 140μg/mL. The synthesis of Plt, however, was not detected in PPM medium and was nearly not influenced by the QscR protein in KMB medium. PCA production was inhibited but Plt biosynthesis was not altered after complementation with qscR gene in trans in the strain of M-18Q. The regulation of qscR gene on PCA production was further confirmed by the analysis of β-galactosidase activities from the translational phzA‘-'lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway. These results indicate that QscR can control PCA production negatively but not Plt production in M-18，and show that QscR functions as a global regulator to differently regulate the synthesis of PCA and Plt on the gene expression level.

Abstract:Plantaricin L-1, an anti-Listeria bacteriocin, was produced by Lactobacillus plantarum and successfully purified by SP-Sepharose FF cation exchange chromatography. The mechanism on energized cells of Listeria monocytogenes was studied with purified plantaricin L-1. After adding plantaricin L-1 to Listeria monocytogenes at 64AU/mL, leakage of intercellular K⁺ ions, inorganic phosphate, lactic dehydrogenase, UV-absorbing materials and the intracellular ATP was observed, and the action resulted in the dissipation of the membrane potential (△ψ) and pH gradient (△pH), two components of the proton motive force (PMF). All the data suggested that the primary site of action of plantaricin L-1 was the cytoplasmic membrane of sensitive cells. By forming the nonselective pores which leak ions and small organic compounds plantaricin L-1 induced the cells death, this action was similar to membrane corruption caused by peptide effect. Penetrability increased due to the enlarged pore and dysfuction of membrane transporters, which ensured efficient killing of target bacteria.

Abstract:Plantaricin L-1, an anti-Listeria bacteriocin, was produced by Lactobacillus plantarum and successfully purified by SP-Sepharose FF cation exchange chromatography. The mechanism on energized cells of Listeria monocytogenes was studied with purified plantaricin L-1. After adding plantaricin L-1 to Listeria monocytogenes at 64AU/mL, leakage of intercellular K⁺ ions, inorganic phosphate, lactic dehydrogenase, UV-absorbing materials and the intracellular ATP was observed, and the action resulted in the dissipation of the membrane potential (△ψ) and pH gradient (△pH), two components of the proton motive force (PMF). All the data suggested that the primary site of action of plantaricin L-1 was the cytoplasmic membrane of sensitive cells. By forming the nonselective pores which leak ions and small organic compounds plantaricin L-1 induced the cells death, this action was similar to membrane corruption caused by peptide effect. Penetrability increased due to the enlarged pore and dysfuction of membrane transporters, which ensured efficient killing of target bacteria.

Abstract:A series of mutants of transglutaminase(TGase) gene from Streptomyces fradiae strains were obtained by low energy N⁺ ion beam irradiation, and the irradiation dose response curve was also investigated. Both the wild type and the mutants of TGase were compared by PCR-SSCP, and specific fragments were sequenced. Then the types of TGase gene mutation induced by N⁺ were characterized. The results show that the types of DNA base mutation included the transition, the transversion and the deletion. In all of the 24 bases of mutants, the base replacement occupies about 87.5% of the total mutants with only a small portion of gene deletion (12.5%). Interestingly, the frequency of the base transition (58.3%) is obviously higher than that of the base transversion (29.2%) among the base replacements. The base substitutions induced by N⁺ ion beam irradiation are C→T transition, A→G transition, G→T transversion, C→G transversion. Furthermore, mutations can be induced in all of the four DNA bases by low energy N⁺ ion beam irradiation, and cytosine shows the highest frequency of mutation.

Abstract:A series of mutants of transglutaminase(TGase) gene from Streptomyces fradiae strains were obtained by low energy N⁺ ion beam irradiation, and the irradiation dose response curve was also investigated. Both the wild type and the mutants of TGase were compared by PCR-SSCP, and specific fragments were sequenced. Then the types of TGase gene mutation induced by N⁺ were characterized. The results show that the types of DNA base mutation included the transition, the transversion and the deletion. In all of the 24 bases of mutants, the base replacement occupies about 87.5% of the total mutants with only a small portion of gene deletion (12.5%). Interestingly, the frequency of the base transition (58.3%) is obviously higher than that of the base transversion (29.2%) among the base replacements. The base substitutions induced by N⁺ ion beam irradiation are C→T transition, A→G transition, G→T transversion, C→G transversion. Furthermore, mutations can be induced in all of the four DNA bases by low energy N⁺ ion beam irradiation, and cytosine shows the highest frequency of mutation.

Abstract:Several spontaneous E. coli mutants with the similar phenotype as that in the condition of amino acid deficiency were obtained on the selective media. One of the mutants (LCH001) showing slow growth phenotype on LB agar plate and pink or white colonies on MacConkey agar plate was mapped at rpoC gene encoding the β′ subunit of RNA polymerase by phage P1 transduction and transformation assays and found to be a new site mutation from G to T at 3406bp in the rpoC gene, which resulted in the amino acid change from Glycine (GGT) to Cysteine (TGT). The effect of the mutation on transcriptional activity of both stringent and non-stringent controlled promoters in vivo was measured by determining the β-galactolactase activity of the growing cells. Results showed that the transcriptional activity of the mutant LCH001 reduced greatly on the stringent promoter, but increased significantly on the non-stringent promoter. The β-galactolactase activity of the mutant LCH001 transcribed on stringent promoter was 18% lower, but 5-fold higher on the non-stringent controlled promoter than that of the wild-type strain CLT5034. This finding may give insights into future studies of the structure-function relationship of RNA polymerase as well as its role in the stringent response of bacteria.

Abstract:Several spontaneous E. coli mutants with the similar phenotype as that in the condition of amino acid deficiency were obtained on the selective media. One of the mutants (LCH001) showing slow growth phenotype on LB agar plate and pink or white colonies on MacConkey agar plate was mapped at rpoC gene encoding the β′ subunit of RNA polymerase by phage P1 transduction and transformation assays and found to be a new site mutation from G to T at 3406bp in the rpoC gene, which resulted in the amino acid change from Glycine (GGT) to Cysteine (TGT). The effect of the mutation on transcriptional activity of both stringent and non-stringent controlled promoters in vivo was measured by determining the β-galactolactase activity of the growing cells. Results showed that the transcriptional activity of the mutant LCH001 reduced greatly on the stringent promoter, but increased significantly on the non-stringent promoter. The β-galactolactase activity of the mutant LCH001 transcribed on stringent promoter was 18% lower, but 5-fold higher on the non-stringent controlled promoter than that of the wild-type strain CLT5034. This finding may give insights into future studies of the structure-function relationship of RNA polymerase as well as its role in the stringent response of bacteria.

Abstract:Ergosterol, the main sterol in yeast, is responsible for structural membrane features such as fluidity and permeability. Additionally, ergosterol is economically important as a precursor of vitamin D₂. The biosynthesis of sterols in yeast is complex. As an enzyme of the later ergosterol biosynthesis, the sterol C-22 desaturase encoded by ERG5 gene is required to form the C-22 (23) double bond in the sterol side chain. In order to know the regulation of C-22 sterol desaturase in the ergosterol biosynthesis, ERG5 gene was cloned and over-expressed in the Saccharomyces cerevisiae. Primer 1 (5′-GTCGGTACCTCCAATGACAATAAATACC-3′, KpnⅠ) and primer 2 (5′-AAGGATCCTAGCAGA TCATTAGCTGTAG-3′, BamHⅠ) were designed according to the ERG5 sequence in GenBank. A 1.8kb DNA fragment containing the open reading frame and terminator of ERG5 gene was amplified from Saccharomyces cerevisiae YSF-20 by PCR and inserted into YEp352 to generate recombinant plasmid pYE5. To express ERG5 gene properly in S. cerevisiae, the recombinant expression plasmid pYPE5 containing ERG5 from pYE5 under the control of PGK1 promoter, the URA3 gene as the selection marker and the plasmid YEp352 as the vector was constructed, and then they were introduced into Saccharomyces cerevisiae YS58. To make sure the plasmid pYPE5 in the YS58 acted properly, the disruptant (YSE5) was created by deleting a 0.4kb fragment of ERG5 gene and inserting the CUP1 gene into the ERG5 and transforming the YS58. And then the disruptant (YSE5) was transformed with the plasmid pYPE5 carrying the corresponding complementing ERG5 gene to control the activity of the over-expressed ERG5 gene and restauration of the wild-type sterol pattern. The sterol profile of the disruptant (YSE5) demonstrated that ergosta-5, 7-dien-3β-ol was accumulated which was very similar to ergosterol but with a saturated side chain. In contrast, the YSE5 (pYPE5) strain contains predominantly ergosterol. The sterol content of the transformant was analyzed using gas chromatography (GC) analysis. The result shows that ergosterol production in recombinant strains was reduced. And the experiment of the effect of culturing time shows that ergosterol productions in recombinant strains were always lower than YS58 (pYPE5) from 24～48h culturing time. Under the optimal culture condition, ergosterol content in recombinant strain YS58 (pYPE5) was about 0.70-fold of that in the referring strain.

Abstract:Ergosterol, the main sterol in yeast, is responsible for structural membrane features such as fluidity and permeability. Additionally, ergosterol is economically important as a precursor of vitamin D₂. The biosynthesis of sterols in yeast is complex. As an enzyme of the later ergosterol biosynthesis, the sterol C-22 desaturase encoded by ERG5 gene is required to form the C-22 (23) double bond in the sterol side chain. In order to know the regulation of C-22 sterol desaturase in the ergosterol biosynthesis, ERG5 gene was cloned and over-expressed in the Saccharomyces cerevisiae. Primer 1 (5′-GTCGGTACCTCCAATGACAATAAATACC-3′, KpnⅠ) and primer 2 (5′-AAGGATCCTAGCAGA TCATTAGCTGTAG-3′, BamHⅠ) were designed according to the ERG5 sequence in GenBank. A 1.8kb DNA fragment containing the open reading frame and terminator of ERG5 gene was amplified from Saccharomyces cerevisiae YSF-20 by PCR and inserted into YEp352 to generate recombinant plasmid pYE5. To express ERG5 gene properly in S. cerevisiae, the recombinant expression plasmid pYPE5 containing ERG5 from pYE5 under the control of PGK1 promoter, the URA3 gene as the selection marker and the plasmid YEp352 as the vector was constructed, and then they were introduced into Saccharomyces cerevisiae YS58. To make sure the plasmid pYPE5 in the YS58 acted properly, the disruptant (YSE5) was created by deleting a 0.4kb fragment of ERG5 gene and inserting the CUP1 gene into the ERG5 and transforming the YS58. And then the disruptant (YSE5) was transformed with the plasmid pYPE5 carrying the corresponding complementing ERG5 gene to control the activity of the over-expressed ERG5 gene and restauration of the wild-type sterol pattern. The sterol profile of the disruptant (YSE5) demonstrated that ergosta-5, 7-dien-3β-ol was accumulated which was very similar to ergosterol but with a saturated side chain. In contrast, the YSE5 (pYPE5) strain contains predominantly ergosterol. The sterol content of the transformant was analyzed using gas chromatography (GC) analysis. The result shows that ergosterol production in recombinant strains was reduced. And the experiment of the effect of culturing time shows that ergosterol productions in recombinant strains were always lower than YS58 (pYPE5) from 24～48h culturing time. Under the optimal culture condition, ergosterol content in recombinant strain YS58 (pYPE5) was about 0.70-fold of that in the referring strain.

Abstract:Co-expression of phytase and mannanase in Pichia pastoris is a useful way to reduce the production cost in feedstuff industry.Based on the published DNA sequences of phytase gene and mannanase gene, primers were designed and genes phyA and man were cloned by PCR from Aspergillus terreus and the plasmid pHBM1201,respectively. Then the two fragments were treated and inserted into the same expression vector pHBM907C, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pHBM907C-phyA and the plasmid pHBM907C-man. The phyA expression cassette was combined to the expression vector pHBM907C-man which contains the expression cassette of mannanase, so pHBM907C-phyA-man was obtained. The recombinant expression plasmid pHBM907C-phyA-man was digested by SalⅠ and introduced into the chromosomes of Pichia pastoris GS115 by using the LiCl/PEG method. Following transformation, several parameters that demonstrated the expression of phytase and mannanase were measured. Firstly, the two different Petri dishes that contain enzymatic substrates such as calcium phytate and mannose were screened simultaneously, thus 100 clones were found to be positive on both of these plates. Secondly, 6 clones among them were chosen for induced expression at shakeflasks showing the probability of high expression. After that, some relative enzymatic properties were measured. At 72 hours′ induction in the condition of shake cultivation, the enzyme activity of phytase in supernatant was 120.6U/mL, while the enzyme activity of mannanase in supernatant was 39.7U/mL. The expression product phytase was active under pH2.0～6.5, and the activity was up to the highest under pH5.5. And the other expression product mannanase was active under pH5.5～10.5,and the activity was up to the highest under pH7.5. The optimal temperatures for the two enzyme were both around 52℃:the optimal temperature for phytase activity was 50℃,and that for mannanase was 55℃.At last, stability test of the engineered yeast was taken, and the engineered yeast still showed an excellent stability even after 10 generation growth in the absence of selective pressure. The stable double functional engineered yeast simultaneously expressing extracellular phytase and mannanase is obtained. It will satisfy the demand for industrialized production in some degree.

Abstract:Co-expression of phytase and mannanase in Pichia pastoris is a useful way to reduce the production cost in feedstuff industry.Based on the published DNA sequences of phytase gene and mannanase gene, primers were designed and genes phyA and man were cloned by PCR from Aspergillus terreus and the plasmid pHBM1201,respectively. Then the two fragments were treated and inserted into the same expression vector pHBM907C, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pHBM907C-phyA and the plasmid pHBM907C-man. The phyA expression cassette was combined to the expression vector pHBM907C-man which contains the expression cassette of mannanase, so pHBM907C-phyA-man was obtained. The recombinant expression plasmid pHBM907C-phyA-man was digested by SalⅠ and introduced into the chromosomes of Pichia pastoris GS115 by using the LiCl/PEG method. Following transformation, several parameters that demonstrated the expression of phytase and mannanase were measured. Firstly, the two different Petri dishes that contain enzymatic substrates such as calcium phytate and mannose were screened simultaneously, thus 100 clones were found to be positive on both of these plates. Secondly, 6 clones among them were chosen for induced expression at shakeflasks showing the probability of high expression. After that, some relative enzymatic properties were measured. At 72 hours′ induction in the condition of shake cultivation, the enzyme activity of phytase in supernatant was 120.6U/mL, while the enzyme activity of mannanase in supernatant was 39.7U/mL. The expression product phytase was active under pH2.0～6.5, and the activity was up to the highest under pH5.5. And the other expression product mannanase was active under pH5.5～10.5,and the activity was up to the highest under pH7.5. The optimal temperatures for the two enzyme were both around 52℃:the optimal temperature for phytase activity was 50℃,and that for mannanase was 55℃.At last, stability test of the engineered yeast was taken, and the engineered yeast still showed an excellent stability even after 10 generation growth in the absence of selective pressure. The stable double functional engineered yeast simultaneously expressing extracellular phytase and mannanase is obtained. It will satisfy the demand for industrialized production in some degree.

Abstract:DNA extraction from the rumen of three species of goat (boer goat, Nanjiang yellow goat, Inner Mongolia cashmere goat) was followed by Polymerase Chain Reaction (PCR) amplification of the beta subunit of the RNA polymerase (rpoB) and 16S rDNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterial community structure. The results showed the rpoB DGGE profiles comprised fewer bands than those of 16S rDNA profiles and were easier to analyze. The gene for rpoB is a single copy gene unlike 16S rDNA. So using the rpoB gene offeres a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. The bacteria community structure of different goats were similar to each other. The similarities within species were noticeably higher than that between species. Goat species were found to influence the rumen microbe community. Phylogenetic and sequence similarity analyses of the resultant 14 clone sequences in16S rDNA DGGE libraries revealed that 4 clone show similarity over 97% with that of database sequences, while the rest present similarity in a range of 89%～96%, and 13 clone of all were similar to those unidentified rumen bacteria. These results suggest that DGGE followed by clone technique is a practicable protocol to research the complex community of rumen microbe.

Abstract:DNA extraction from the rumen of three species of goat (boer goat, Nanjiang yellow goat, Inner Mongolia cashmere goat) was followed by Polymerase Chain Reaction (PCR) amplification of the beta subunit of the RNA polymerase (rpoB) and 16S rDNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterial community structure. The results showed the rpoB DGGE profiles comprised fewer bands than those of 16S rDNA profiles and were easier to analyze. The gene for rpoB is a single copy gene unlike 16S rDNA. So using the rpoB gene offeres a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. The bacteria community structure of different goats were similar to each other. The similarities within species were noticeably higher than that between species. Goat species were found to influence the rumen microbe community. Phylogenetic and sequence similarity analyses of the resultant 14 clone sequences in16S rDNA DGGE libraries revealed that 4 clone show similarity over 97% with that of database sequences, while the rest present similarity in a range of 89%～96%, and 13 clone of all were similar to those unidentified rumen bacteria. These results suggest that DGGE followed by clone technique is a practicable protocol to research the complex community of rumen microbe.

Abstract:Recent investigations on the microbial ecology of oil reservoirs in a variety of locales indicated that these habitats harbor various assemblages. In this study, a cultured-independent molecular technique, Terminal Restriction Fragment Length Polymorphism (T-RFLP), was used to analyze the microbial diversity of an injection well (S12-ZHU) and three related production wells (S12-4、S12-5 and S12-19) in the ShengLi oilfield (Shandong province, China). The 16S rRNA genes were amplified by PCR with the 5′carboxy-fluorescein (5-FAM)-labelled universal forward primers (27F for bacteria and 21F for archaea) and a universal reverse primer (1495R). Then the 16S rRNA genes were digested with restriction enzymes (HaeⅢ and HhaⅠ) and analyzed by using an automated DNA sequencer. The Shannon-Wiener Diversity index, based on the T-RFLP profiles, indicated that the bacterial and archaeal species richness in the injection well was higher than those of the production ones. The similarity coefficient showed the microbial community similarity among the four samples was 22.4%～30.8% (Bacteria) and 20.8%～34.5％ (Archaea), respectively. According to the analysis by TAP T-RFLP program, species belonging to Pseudomonas, Marinobacter and Methanosarcina as well as some uncultured archaeon were supposed to be the dominant bacteria in all four samples. Thus, this study indicates that T-RFLP is useful for analysis of the microbial diversity in petroleum reservoirs.

Abstract:Recent investigations on the microbial ecology of oil reservoirs in a variety of locales indicated that these habitats harbor various assemblages. In this study, a cultured-independent molecular technique, Terminal Restriction Fragment Length Polymorphism (T-RFLP), was used to analyze the microbial diversity of an injection well (S12-ZHU) and three related production wells (S12-4、S12-5 and S12-19) in the ShengLi oilfield (Shandong province, China). The 16S rRNA genes were amplified by PCR with the 5′carboxy-fluorescein (5-FAM)-labelled universal forward primers (27F for bacteria and 21F for archaea) and a universal reverse primer (1495R). Then the 16S rRNA genes were digested with restriction enzymes (HaeⅢ and HhaⅠ) and analyzed by using an automated DNA sequencer. The Shannon-Wiener Diversity index, based on the T-RFLP profiles, indicated that the bacterial and archaeal species richness in the injection well was higher than those of the production ones. The similarity coefficient showed the microbial community similarity among the four samples was 22.4%～30.8% (Bacteria) and 20.8%～34.5％ (Archaea), respectively. According to the analysis by TAP T-RFLP program, species belonging to Pseudomonas, Marinobacter and Methanosarcina as well as some uncultured archaeon were supposed to be the dominant bacteria in all four samples. Thus, this study indicates that T-RFLP is useful for analysis of the microbial diversity in petroleum reservoirs.

Abstract:The prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China was investigated using PCR-DGGE and rRNA approaches. Total community DNA was extracted and purified by a direct method, which yielded amplified DNA of high molecular weight for samples. A variable region of 16S rRNA gene was then amplified by PCR with bacterial and archaeal primers and analyzed by denaturing gradient gel electrophoresis (DGGE). Twenty-seven major bands were detected in the bacterial DGGE profile of the sample, but only one band of pure culture strains of bacteria isolated from the Kunming Salt Mine matched with one band of sample. No band of pure culture strains of archaea isolated from the Kunming Salt Mine matched with 18 major bands of sample. The results indicated that most of microbes in this environment are likely uncultivable. Clones on the plate were not the predominant species in the community. Two 16S rRNA gene clone libraries (bacteria and archaea) were also constructed, and 36 and 20 clones were selected for amplified ribosomal DNA restriction analysis (ARDRA). ARDRA with enzymes AfaⅠ、HhaⅠ、HaeⅢ revealed 10 bacterial operational taxonomic units (OTUs), with three most abundant OTUs accounting for 38.9%, 25.0%, 16.7% of all the bacterial 16S rDNA clones, respectively. The remaining 7 OTUs presented at low levels, were represented by a single clone. Eight archaeal OTUs were obtained but no predominant OTUs. Some clones were sequenced and each sequence was compared with all nucleotide sequences in GenBank database. Examination of 16S rDNA clones showed that the ancient salt deposits in the Kunming Salt Mine contained a phylogenetically diverse population of organisms from the Bacteria domain with members of three major lineages represented: α-proteobacteria, γ-Proteobacteria and Actinobacteria, especially Pseudomonas. Surprisingly, we recovered a variety of sequence closely related to Actinobacteria which was not found in other salt deposits. All of archaeal clones are from Halorubrum, Haloterrigena and uncultured archaea. The results of DGGE and clone library profiling analysis both indicated that microbial community of the Kunming Salt Mine had higher diversity. In this initial survey, our polyphasic approaches demonstrated that novel and uncultured microbes thrive in the ancient salt deposits of the Kunming Salt Mine. Molecular analysis of the microbial diversity in salt deposits provides foundation for better application of microbial resources.

Abstract:The prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China was investigated using PCR-DGGE and rRNA approaches. Total community DNA was extracted and purified by a direct method, which yielded amplified DNA of high molecular weight for samples. A variable region of 16S rRNA gene was then amplified by PCR with bacterial and archaeal primers and analyzed by denaturing gradient gel electrophoresis (DGGE). Twenty-seven major bands were detected in the bacterial DGGE profile of the sample, but only one band of pure culture strains of bacteria isolated from the Kunming Salt Mine matched with one band of sample. No band of pure culture strains of archaea isolated from the Kunming Salt Mine matched with 18 major bands of sample. The results indicated that most of microbes in this environment are likely uncultivable. Clones on the plate were not the predominant species in the community. Two 16S rRNA gene clone libraries (bacteria and archaea) were also constructed, and 36 and 20 clones were selected for amplified ribosomal DNA restriction analysis (ARDRA). ARDRA with enzymes AfaⅠ、HhaⅠ、HaeⅢ revealed 10 bacterial operational taxonomic units (OTUs), with three most abundant OTUs accounting for 38.9%, 25.0%, 16.7% of all the bacterial 16S rDNA clones, respectively. The remaining 7 OTUs presented at low levels, were represented by a single clone. Eight archaeal OTUs were obtained but no predominant OTUs. Some clones were sequenced and each sequence was compared with all nucleotide sequences in GenBank database. Examination of 16S rDNA clones showed that the ancient salt deposits in the Kunming Salt Mine contained a phylogenetically diverse population of organisms from the Bacteria domain with members of three major lineages represented: α-proteobacteria, γ-Proteobacteria and Actinobacteria, especially Pseudomonas. Surprisingly, we recovered a variety of sequence closely related to Actinobacteria which was not found in other salt deposits. All of archaeal clones are from Halorubrum, Haloterrigena and uncultured archaea. The results of DGGE and clone library profiling analysis both indicated that microbial community of the Kunming Salt Mine had higher diversity. In this initial survey, our polyphasic approaches demonstrated that novel and uncultured microbes thrive in the ancient salt deposits of the Kunming Salt Mine. Molecular analysis of the microbial diversity in salt deposits provides foundation for better application of microbial resources.

Abstract:Molecular analysis of community structure can help diagnose problems in malfunctioning full-scale wastewater treatment system. A lab-scale A₁-A₂-O fixed biofilm system with highly efficient nitrification (NH₃-N removal at 95.2%) was set up as a reference for an industrial system with poor performance of nitrification (NH₃-N removal efficiency at -106%) for treating coking wastewater. Composition of nitrifying bacteria of biofilm samples in aerobic reactors of the two systems was compared by 16S rDNA clone library analysis. The composition of clone library of the lab-scale system indicate Nitrosomonas europaea-Nitrosoccus mobilis cluster and sublineage Ⅰ of Nitrospira genus are dominant ammonia and nitrite oxidizers in this process respectively. However, there were no clones related to nitrifying bacteria in clone library of the industrial system which suggested low abundance of nitrifying bacteria in this system. Further, Real-Time PCR with Taqman probe revealed that 16S rDNA copy number of Nitrospira in aerobic reactor of the lab-scale system was 3.4×10^{6/μg DNA of biofilm which is 300 times higher than that in the industrial system. The absence of Nitrosomonas and Nitrospira in the industrial system leads to poor performance of nitrification. Building up of a high population level of Nitrosomonas and Nitrospira in aerobic reactor is the key to improve efficiency of nitrification of the industrial system.}

Abstract:Molecular analysis of community structure can help diagnose problems in malfunctioning full-scale wastewater treatment system. A lab-scale A₁-A₂-O fixed biofilm system with highly efficient nitrification (NH₃-N removal at 95.2%) was set up as a reference for an industrial system with poor performance of nitrification (NH₃-N removal efficiency at -106%) for treating coking wastewater. Composition of nitrifying bacteria of biofilm samples in aerobic reactors of the two systems was compared by 16S rDNA clone library analysis. The composition of clone library of the lab-scale system indicate Nitrosomonas europaea-Nitrosoccus mobilis cluster and sublineage Ⅰ of Nitrospira genus are dominant ammonia and nitrite oxidizers in this process respectively. However, there were no clones related to nitrifying bacteria in clone library of the industrial system which suggested low abundance of nitrifying bacteria in this system. Further, Real-Time PCR with Taqman probe revealed that 16S rDNA copy number of Nitrospira in aerobic reactor of the lab-scale system was 3.4×10^{6/μg DNA of biofilm which is 300 times higher than that in the industrial system. The absence of Nitrosomonas and Nitrospira in the industrial system leads to poor performance of nitrification. Building up of a high population level of Nitrosomonas and Nitrospira in aerobic reactor is the key to improve efficiency of nitrification of the industrial system.}

Abstract:A quantitative competitive PCR (QC-PCR) system was developed to quantify the number and analyze the function of the Rhodococcus ruber Em1 strain in a wastewater treatment system in Nanchong oil refinery plant. Strain Em1 was able to degrade various kinds of hydrocarbons and aromatic compounds with high efficiency and produce bio-emulsifier, so it was introduced into the waste liquid petroleum-disposing system. The sediment samples were collected from the disposing system in the range of 5 months, and then the numbers of strain Em1 and degrading efficiencies were studied. The results showed that the primers based on 16S rRNA gene sequence of strain Em1 were specific at species level. The PCR products amplified from sediment total DNA with the specific primers were cloned and sequenced, in which 62.2% were the fragments of 16S rRNA gene of strain Em1. Furthermore, the number of Em1 strain ranging from 3.4×10⁵～4.3×10⁸CFU/g in the sediment samples were detected, which indicated that the strain Em1 added into purposely did exist stably and reproduced well in the waste-deposing system during a long period. The high relativity, with relative coefficient R² of 0.89, between Em1 cell number and the amount of COD (Chemical Oxygen Demand) removal proved that the strain Em1 played an important role in this bio-augmentation treatment system.

Abstract:A quantitative competitive PCR (QC-PCR) system was developed to quantify the number and analyze the function of the Rhodococcus ruber Em1 strain in a wastewater treatment system in Nanchong oil refinery plant. Strain Em1 was able to degrade various kinds of hydrocarbons and aromatic compounds with high efficiency and produce bio-emulsifier, so it was introduced into the waste liquid petroleum-disposing system. The sediment samples were collected from the disposing system in the range of 5 months, and then the numbers of strain Em1 and degrading efficiencies were studied. The results showed that the primers based on 16S rRNA gene sequence of strain Em1 were specific at species level. The PCR products amplified from sediment total DNA with the specific primers were cloned and sequenced, in which 62.2% were the fragments of 16S rRNA gene of strain Em1. Furthermore, the number of Em1 strain ranging from 3.4×10⁵～4.3×10⁸CFU/g in the sediment samples were detected, which indicated that the strain Em1 added into purposely did exist stably and reproduced well in the waste-deposing system during a long period. The high relativity, with relative coefficient R² of 0.89, between Em1 cell number and the amount of COD (Chemical Oxygen Demand) removal proved that the strain Em1 played an important role in this bio-augmentation treatment system.

Abstract:In order to investigate the potential influence of the organic pollutants on the microbial composition and diversity, terminal restriction fragment length polymorphism (tRFLP) and 16S ribosomal DNA(rDNA) clone libraries combined with water quality analysis were selected to compare the structure of bacterial communities in four water bodies contaminated by different degrees of organic matter. tRFLP profiles of the waters and sediments all showed complex patterns and high similarity, however, some certain populations enriched with the pollution enhanced. Especially, the similarity of communities accorded strictly with that of water quality. Principal component analysis (PCA) indicated that the terminal restriction fragments (TRF) of bacteria in the waters and sediments grouped into different clusters. 16S ribosomal DNA sequences in the Songhua River sediment fell into ten known phyla and Proteobacteria are predominant with 21.92% of clones (in which the β-Proteobacteria accounts for 10.96%). The sediment seriously polluted by domestic wastewater comprised of only 7 phyla, in which Proteobacteria was predominant with 47.37% of clones (subdivisions α-Proteobacteria and δ/ε-Proteobacteria were 21.05% and 15.79%, respectively). This study demonstrated that the long-term drainage of organic wastewater reduced the microbial diversity, and some functional microbes that are responsible for the degradation of organic matter, became dominant.

Abstract:In order to investigate the potential influence of the organic pollutants on the microbial composition and diversity, terminal restriction fragment length polymorphism (tRFLP) and 16S ribosomal DNA(rDNA) clone libraries combined with water quality analysis were selected to compare the structure of bacterial communities in four water bodies contaminated by different degrees of organic matter. tRFLP profiles of the waters and sediments all showed complex patterns and high similarity, however, some certain populations enriched with the pollution enhanced. Especially, the similarity of communities accorded strictly with that of water quality. Principal component analysis (PCA) indicated that the terminal restriction fragments (TRF) of bacteria in the waters and sediments grouped into different clusters. 16S ribosomal DNA sequences in the Songhua River sediment fell into ten known phyla and Proteobacteria are predominant with 21.92% of clones (in which the β-Proteobacteria accounts for 10.96%). The sediment seriously polluted by domestic wastewater comprised of only 7 phyla, in which Proteobacteria was predominant with 47.37% of clones (subdivisions α-Proteobacteria and δ/ε-Proteobacteria were 21.05% and 15.79%, respectively). This study demonstrated that the long-term drainage of organic wastewater reduced the microbial diversity, and some functional microbes that are responsible for the degradation of organic matter, became dominant.

Abstract:The β domains of autotransporters (also called type Ⅴ secretion system) have been demonstrated to be feasible tools to display foreign passenger domain on the bacterial surface. In the present work, using the DNA manipulation the type Ⅴ secretion system MisL displaying the binding doamin FedF1 of the F18ab fimbrial adhesion was constructed, and the full length adhesin FedF of F18ab fimbriae and the FedF mutant (with the 88th and 89th amino acid residues changed from Histadine to Alamine) on the surface of E. coli DH5α, respectively. The recombinant E. coli DH5α with the different recombinant plasmids of pnirBMisL-fedF1, pnirBMisL-fedF and pnirBMisL-fedF(M) were induced at anaerobic conditions. After induction, the binding doamin FedF1 of the FedF and adhesin FedF displayed on the surface of E. coli DH5α were tested for their agglutination and adhesion capability with the anti-rabbit sera against FedF subunit or the small intestinal epithelial cells from susceptible piglets. The results showed that the pnirBMisL-fedF1 or pnirBMisL-fedF recombinant bacteria could agglutinate with the anti-rabbit sera against FedF subunit and adhere to the small intestinal epithelial cells well. But the recombinant bacterial strain with the recombinant plasmid pnirBMisL-fedF(M) completely abolished the agglutination characteristic and receptor adhesiveness. These results confirmed that the binding doamin FedF1 of the F18ab fimbrial adhesin and the adhesin FedF of F18ab fimbriae could be transported and displayed functionally on the surface of E. coli DH5α by using type Ⅴ secretion system and the His-88 and His-89 amino acid residues located in the FedF adhesin were important for the formation of the binding domain of the adhesin FedF of F18ab fimbriae.

Abstract:The β domains of autotransporters (also called type Ⅴ secretion system) have been demonstrated to be feasible tools to display foreign passenger domain on the bacterial surface. In the present work, using the DNA manipulation the type Ⅴ secretion system MisL displaying the binding doamin FedF1 of the F18ab fimbrial adhesion was constructed, and the full length adhesin FedF of F18ab fimbriae and the FedF mutant (with the 88th and 89th amino acid residues changed from Histadine to Alamine) on the surface of E. coli DH5α, respectively. The recombinant E. coli DH5α with the different recombinant plasmids of pnirBMisL-fedF1, pnirBMisL-fedF and pnirBMisL-fedF(M) were induced at anaerobic conditions. After induction, the binding doamin FedF1 of the FedF and adhesin FedF displayed on the surface of E. coli DH5α were tested for their agglutination and adhesion capability with the anti-rabbit sera against FedF subunit or the small intestinal epithelial cells from susceptible piglets. The results showed that the pnirBMisL-fedF1 or pnirBMisL-fedF recombinant bacteria could agglutinate with the anti-rabbit sera against FedF subunit and adhere to the small intestinal epithelial cells well. But the recombinant bacterial strain with the recombinant plasmid pnirBMisL-fedF(M) completely abolished the agglutination characteristic and receptor adhesiveness. These results confirmed that the binding doamin FedF1 of the F18ab fimbrial adhesin and the adhesin FedF of F18ab fimbriae could be transported and displayed functionally on the surface of E. coli DH5α by using type Ⅴ secretion system and the His-88 and His-89 amino acid residues located in the FedF adhesin were important for the formation of the binding domain of the adhesin FedF of F18ab fimbriae.

Abstract:Glycoprotein S1 was the major protein to determine infection and immunogenicity of Infectious bronchitis virus（IBV）. The S1 glycoprotein gene of IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and proved to be S1 gene by sequencing. The E. coli-mycobacterium expression shuttle plasmid pR-α-S1 was constructed by inserting the S1 gene to the pRR3 with human mycobacterium tuberculosis HSP70 promoter and α signal peptide. Then the plasmid pR-α-S1 was introduced into mycobacterium bovis BCG by electroporation to construct a recombinate strain rBCG-S1. The S1 protein could be highly expressed in M. smegmatis mc² 155 when induced by heating and was detected by ELISA and Western blot assays using monoclonal antibody against S1 glycoprotein of IBV. 6 week-old SPF chicken were subcutaneously immunized with 10⁶ cfu rBCG-S1 and each chick was immunized three times at 3 week intervals with the same antigen used for the primary immunization. The protective immunity of rBCG-S1 was identified in vaccinated chickens. Results from the protection test showed the two immunizations with rBCG-S1 could provide protection for chickens from the challenge with virulent nephropathogenic IBV strain X. Haemagglutination inhibition titers were also increased in chickens immunized with the expressed rBCG-S1, and significantly higher titers were detected after challenge. These data indicate that the rBCG-S1 could be used as candidate of a live vector vaccine for NIBV.

Abstract:Glycoprotein S1 was the major protein to determine infection and immunogenicity of Infectious bronchitis virus（IBV）. The S1 glycoprotein gene of IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and proved to be S1 gene by sequencing. The E. coli-mycobacterium expression shuttle plasmid pR-α-S1 was constructed by inserting the S1 gene to the pRR3 with human mycobacterium tuberculosis HSP70 promoter and α signal peptide. Then the plasmid pR-α-S1 was introduced into mycobacterium bovis BCG by electroporation to construct a recombinate strain rBCG-S1. The S1 protein could be highly expressed in M. smegmatis mc² 155 when induced by heating and was detected by ELISA and Western blot assays using monoclonal antibody against S1 glycoprotein of IBV. 6 week-old SPF chicken were subcutaneously immunized with 10⁶ cfu rBCG-S1 and each chick was immunized three times at 3 week intervals with the same antigen used for the primary immunization. The protective immunity of rBCG-S1 was identified in vaccinated chickens. Results from the protection test showed the two immunizations with rBCG-S1 could provide protection for chickens from the challenge with virulent nephropathogenic IBV strain X. Haemagglutination inhibition titers were also increased in chickens immunized with the expressed rBCG-S1, and significantly higher titers were detected after challenge. These data indicate that the rBCG-S1 could be used as candidate of a live vector vaccine for NIBV.

Abstract:The major aim of this study is to determine the location on outer envelope of the genus-specific antigen OmpL1s of Leptospira interrogans, and the inducement of naturally antibody response and types of the antigen, which will offer the evidences to use OmpL1s as the antigen candidate for developing universal genetic engineering vaccine and detection kit. The serum samples from 156 leptospirosis patients in Sichuan area were detected using microscope agglutination test (MAT). By using PCR plus nucleotide sequence analysis, the genotypes of the dominant L. interrogans serogroups in China were demonstrated. Routine genetic engineering technique was applied to construct the prokaryotic expression systems of genotypes ompL1/1 and ompL1/2, and Ni-NTA affinity chromatography was performed to extract the target recombinant products rOmpL1/1 and rOmpL1/2. Immune aurosol electron microscopy was selected to locate the position of OmpL1s on leptospiral envelope. ELISAs based on rOmpL1s were established to confirm the level and types of specific antibody. The results indicated that L. interrogans serogroup Icterohaemorrhagiae remains to be the most important dominant leptospiral serogroup in Sichuan area. There are two ompL1 genotypes of ompL1/1 and ompL1/2 in the dominant leptospiral serogroup in China. And remarkable differences of the nucleotide and putative amino acid sequence similarities between the two genotypes are present. OmpL1s are the protein molecular that located on the external surface of leptospiral envelope. In the 156 cases of leptospirosis patients' serum samples using different dilutions, the positive rates for rOmpL1/1 or rOmpL1/2 specific IgM are 67.9%～79.5% and 75.0%～75.6%, while for for rOmpL1/1 or rOmpL1/2 specific IgG are 71.8%～79.5% and 75.0%～76.9%, respectively. All the results mentioned above lead to the conclusions that OmpL1s is the leptospiral genus-specific superficial protein antigen of L. interrogans and both rOmpL1/1 and rOmpL1/2 can induce humoral response in individuals naturally infected with L. interrogans as well as produce two serum antibodies IgM and IgG with extensive antigenic-cross reaction. Therefore, rOmpL1/1 and rOmpL1/2 can be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

Abstract:The major aim of this study is to determine the location on outer envelope of the genus-specific antigen OmpL1s of Leptospira interrogans, and the inducement of naturally antibody response and types of the antigen, which will offer the evidences to use OmpL1s as the antigen candidate for developing universal genetic engineering vaccine and detection kit. The serum samples from 156 leptospirosis patients in Sichuan area were detected using microscope agglutination test (MAT). By using PCR plus nucleotide sequence analysis, the genotypes of the dominant L. interrogans serogroups in China were demonstrated. Routine genetic engineering technique was applied to construct the prokaryotic expression systems of genotypes ompL1/1 and ompL1/2, and Ni-NTA affinity chromatography was performed to extract the target recombinant products rOmpL1/1 and rOmpL1/2. Immune aurosol electron microscopy was selected to locate the position of OmpL1s on leptospiral envelope. ELISAs based on rOmpL1s were established to confirm the level and types of specific antibody. The results indicated that L. interrogans serogroup Icterohaemorrhagiae remains to be the most important dominant leptospiral serogroup in Sichuan area. There are two ompL1 genotypes of ompL1/1 and ompL1/2 in the dominant leptospiral serogroup in China. And remarkable differences of the nucleotide and putative amino acid sequence similarities between the two genotypes are present. OmpL1s are the protein molecular that located on the external surface of leptospiral envelope. In the 156 cases of leptospirosis patients' serum samples using different dilutions, the positive rates for rOmpL1/1 or rOmpL1/2 specific IgM are 67.9%～79.5% and 75.0%～75.6%, while for for rOmpL1/1 or rOmpL1/2 specific IgG are 71.8%～79.5% and 75.0%～76.9%, respectively. All the results mentioned above lead to the conclusions that OmpL1s is the leptospiral genus-specific superficial protein antigen of L. interrogans and both rOmpL1/1 and rOmpL1/2 can induce humoral response in individuals naturally infected with L. interrogans as well as produce two serum antibodies IgM and IgG with extensive antigenic-cross reaction. Therefore, rOmpL1/1 and rOmpL1/2 can be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

Abstract:Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-ΔE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-ΔE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-ΔE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-ΔE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXΔE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-ΔE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-ΔE3-Rgp can generate typical CPE of CAV-2. CAV2-ΔE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-ΔE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-ΔE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.

Abstract:Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-ΔE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-ΔE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-ΔE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-ΔE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXΔE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-ΔE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-ΔE3-Rgp can generate typical CPE of CAV-2. CAV2-ΔE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-ΔE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-ΔE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.

Abstract:Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100%. The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.

Abstract:Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100%. The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.

Abstract:To develop investigate the recombinant MVA(rMVA) vaccines against PRRSV infection, the ORF4, ORF5 and ORF6 of PRRSV NJ-a strain were subcloned into the MVA transfer vector pⅡLR and the resultant recombinant vector was called pⅡLR-ORF4/ORF5/ORF6. The rMVA was generated by transfecting MVA-infected BHK-21 cells with the recombinant vector and screened by plaque purification after X-gal staining. After six rounds of purification, insertion of PRRSV GP4, GP5 and M genes into the MVA genome was confirmed by PCR analysis and expression of the three proteins was identified by Western-blot and IFA. Each of the tested mice was inoculated with 5×10⁵TCID₅₀/mouse of the rMVA-GP4/GP5/M and boosted 3 weeks later. Neutralization assay showed that PRRSV-specific neutralizing antibodies were detectable at 3 weeks and reached the highest titer (2⁵) by 8 weeks after the primary vaccination, which maintained stable until the end of the experiment. The significant lymphocyte proliferation responses were also observed in mice immunized with rMVA-GP4/GP5/M. These results indicate the rMVA co-expressing PRRSV ORF4, ORF5 andORF6 genes may be an attractive candidate vaccine for preventing PRRSV infection.

Abstract:To develop investigate the recombinant MVA(rMVA) vaccines against PRRSV infection, the ORF4, ORF5 and ORF6 of PRRSV NJ-a strain were subcloned into the MVA transfer vector pⅡLR and the resultant recombinant vector was called pⅡLR-ORF4/ORF5/ORF6. The rMVA was generated by transfecting MVA-infected BHK-21 cells with the recombinant vector and screened by plaque purification after X-gal staining. After six rounds of purification, insertion of PRRSV GP4, GP5 and M genes into the MVA genome was confirmed by PCR analysis and expression of the three proteins was identified by Western-blot and IFA. Each of the tested mice was inoculated with 5×10⁵TCID₅₀/mouse of the rMVA-GP4/GP5/M and boosted 3 weeks later. Neutralization assay showed that PRRSV-specific neutralizing antibodies were detectable at 3 weeks and reached the highest titer (2⁵) by 8 weeks after the primary vaccination, which maintained stable until the end of the experiment. The significant lymphocyte proliferation responses were also observed in mice immunized with rMVA-GP4/GP5/M. These results indicate the rMVA co-expressing PRRSV ORF4, ORF5 andORF6 genes may be an attractive candidate vaccine for preventing PRRSV infection.

Abstract:An acidophilic, aerobic and chemoheterotrophic bacterial strain Teng-A was isolated from acidic environmental samples collected at sulfidic hot springs of Tengchong County, Yunnan Province, China. Cells of strain Teng-A was rod-shaped (0.6～0.8 μm×1.0～1.5 μm), Gram-negative, motile with flagella. Strain Teng-A grew well at temperature of 29～33oC and at pH of 3.0～4.0. It used a wide variety of organic compounds for growth, but did not use ferrous iron, elemental sulfur, thiosulfate and tetrathionate as the sole energy source. Its G+C content was determined to be 69.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to species of Acidiphilium. Under anoxic conditions, the strain Teng-A reduced Fe(Ⅲ)to Fe(Ⅱ) with glucose or hy drogen as electron donor (reduction rate is 11.56mg/L·day and 15.34mg/L·day, respectively). Metabolisms/Oxidation of ferrous iron by Acidithiobacillus ferrooxidans LJ-1 and Leptospirilum ferriphilum LJ-2, in the presence and absence of strain Teng-A were studied. When incubated with strain Teng-A, the oxidation rates of Fe(Ⅱ) was slightly decreased at the first 3 days (0.44g/L·day and 0.41g/L·day respectively) compared to pure culture of At ferrooxidans and L. ferriphilum, but all Fe(Ⅱ) was completely oxidized after 5 days. It was found that the morphologies of precipitates of Fe(Ⅲ) produced during pure and mixed cultivation were different. The potential application of Acidiphilium in bioleaching and its potential role during formation of precipitated ores were discussed.

Abstract:An acidophilic, aerobic and chemoheterotrophic bacterial strain Teng-A was isolated from acidic environmental samples collected at sulfidic hot springs of Tengchong County, Yunnan Province, China. Cells of strain Teng-A was rod-shaped (0.6～0.8 μm×1.0～1.5 μm), Gram-negative, motile with flagella. Strain Teng-A grew well at temperature of 29～33oC and at pH of 3.0～4.0. It used a wide variety of organic compounds for growth, but did not use ferrous iron, elemental sulfur, thiosulfate and tetrathionate as the sole energy source. Its G+C content was determined to be 69.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to species of Acidiphilium. Under anoxic conditions, the strain Teng-A reduced Fe(Ⅲ)to Fe(Ⅱ) with glucose or hy drogen as electron donor (reduction rate is 11.56mg/L·day and 15.34mg/L·day, respectively). Metabolisms/Oxidation of ferrous iron by Acidithiobacillus ferrooxidans LJ-1 and Leptospirilum ferriphilum LJ-2, in the presence and absence of strain Teng-A were studied. When incubated with strain Teng-A, the oxidation rates of Fe(Ⅱ) was slightly decreased at the first 3 days (0.44g/L·day and 0.41g/L·day respectively) compared to pure culture of At ferrooxidans and L. ferriphilum, but all Fe(Ⅱ) was completely oxidized after 5 days. It was found that the morphologies of precipitates of Fe(Ⅲ) produced during pure and mixed cultivation were different. The potential application of Acidiphilium in bioleaching and its potential role during formation of precipitated ores were discussed.

Abstract:In the present study, a AHL-utilizing strain R1 was isolated and identified as the genus Rhodosporidium toruloides R1 by physi-biochemical approaches and 18S rDNA sequence analysis, and this strain was designated as R. toruloides R1. Results showed that R. toruloides R1 exhibited the ability to utilize and degrade the all N-acyl homoserine lactones tested in this study. Coculture of R. toruloides R1 with Erwinia carotovora subsp. Carotovora effectively inhibit the soft rot disease of patato caused by E. carotovora. To the best of our knowledge, this is the first report on AHL-degradation of yeast cells.

Abstract:In the present study, a AHL-utilizing strain R1 was isolated and identified as the genus Rhodosporidium toruloides R1 by physi-biochemical approaches and 18S rDNA sequence analysis, and this strain was designated as R. toruloides R1. Results showed that R. toruloides R1 exhibited the ability to utilize and degrade the all N-acyl homoserine lactones tested in this study. Coculture of R. toruloides R1 with Erwinia carotovora subsp. Carotovora effectively inhibit the soft rot disease of patato caused by E. carotovora. To the best of our knowledge, this is the first report on AHL-degradation of yeast cells.

Abstract:The 26S rRNA gene D1/D2 domain sequences of 15 strains originally identified as Galactomyces geotrichum from the Chinese Industry Culture Collection (CICC) were determined. The results indicated that these strains differed from the type strain of Galactomyces geotrichum and other species of the genus remarkably. Two groups were recognized from the 15 strains which possibly represent 2 novel species of Galactomyces. Further molecular study is needed to confirm their taxonomic status.

Abstract:The 26S rRNA gene D1/D2 domain sequences of 15 strains originally identified as Galactomyces geotrichum from the Chinese Industry Culture Collection (CICC) were determined. The results indicated that these strains differed from the type strain of Galactomyces geotrichum and other species of the genus remarkably. Two groups were recognized from the 15 strains which possibly represent 2 novel species of Galactomyces. Further molecular study is needed to confirm their taxonomic status.

Abstract:A moderately halophilic bacterium Halomonas sp. NJ223 was isolated from Antarctica deep-sea sediment. This bacterium accumulates ectoine as the main compatible solute in response to severe osmotic stress. Ecoine synthase catalyzes circulation of γ-N-acetyl-α，γ-diaminobutyric acid (ADABA) to ectoine in the last step of the three enzymatic steps. The gene of ectoine synthase from this strain was amplified by PCR and the DNA sequence of a 393-bp segment was sequenced. The amino acid sequences of this enzyme present high homology to the known sequence. The significance of this gene was proved by the expression in Escherichia coli. Thus, the amplified fragment was cloned into the expression vector pET-his. The insert position, the size and the reading frame were identified by PCR, restriction digestion and the sequence analysis of the recombinant plasmids. SDS-PAGE shows that the relative molecular mass of the expression product was 15kDa as predicted, which indicated that the recombinant plasmids could express the gene of ectoine synthase. The biosynthetic pathway of ectoine was partially elucidated by renaturation and enzyme activity detection of purified ectoine synthase in vitro. Determination of effect of pH and temperature on enzyme activity shows that the optimal reaction condition of pH was 8.0 and temperature was 25℃.

Abstract:A moderately halophilic bacterium Halomonas sp. NJ223 was isolated from Antarctica deep-sea sediment. This bacterium accumulates ectoine as the main compatible solute in response to severe osmotic stress. Ecoine synthase catalyzes circulation of γ-N-acetyl-α，γ-diaminobutyric acid (ADABA) to ectoine in the last step of the three enzymatic steps. The gene of ectoine synthase from this strain was amplified by PCR and the DNA sequence of a 393-bp segment was sequenced. The amino acid sequences of this enzyme present high homology to the known sequence. The significance of this gene was proved by the expression in Escherichia coli. Thus, the amplified fragment was cloned into the expression vector pET-his. The insert position, the size and the reading frame were identified by PCR, restriction digestion and the sequence analysis of the recombinant plasmids. SDS-PAGE shows that the relative molecular mass of the expression product was 15kDa as predicted, which indicated that the recombinant plasmids could express the gene of ectoine synthase. The biosynthetic pathway of ectoine was partially elucidated by renaturation and enzyme activity detection of purified ectoine synthase in vitro. Determination of effect of pH and temperature on enzyme activity shows that the optimal reaction condition of pH was 8.0 and temperature was 25℃.

Abstract:Candida albicans is the most common kind of human opportunistic pathogen and gets many attentions in recent years. A lot of research has been done. The sites this organism colonizes differ in pH. C. albicans must be able to response to the environmental changes for its survival and pathogenicity. Extracellular environment, especially pH changes，is critical for C. albicans morphological differentiation and virulence. RIM101 pathway is a conserved fungal signal transduction pathway and at least partly controls the cellular pH response through the activity of the zinc finger transcription factor Rim101p. This review concisely summarized recent research on RIM101 pathway, pH response and their relationship in C. albicans.

Abstract:Candida albicans is the most common kind of human opportunistic pathogen and gets many attentions in recent years. A lot of research has been done. The sites this organism colonizes differ in pH. C. albicans must be able to response to the environmental changes for its survival and pathogenicity. Extracellular environment, especially pH changes，is critical for C. albicans morphological differentiation and virulence. RIM101 pathway is a conserved fungal signal transduction pathway and at least partly controls the cellular pH response through the activity of the zinc finger transcription factor Rim101p. This review concisely summarized recent research on RIM101 pathway, pH response and their relationship in C. albicans.

Abstract:Repeat sequence exists in almost all organism genomes. Enterobacterial Repetitive Intergenic Consensus (ERIC), also named Intergenic Repetitive Unit (IRU), is a kind of intergenic repetitive sequence that exists predominantly in Enterobacteria. ERIC(IRU) was firstly discovered in Escherichia coli, followed by identification in many other bacteria. ERIC(IRU) is 127bp long, and some with inserted sequences. Most ERIC(IRU) can be transcribed, and mRNA forms a stem-loop structure. ERIC(IRU) is restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames(ORF). ERIC(IRU) probably modulates the expression of flanking genes. ERIC(IRU) is highly conserved, for either its variation is restricted by the natural selection pressure, or it's “selfish DNA". ERIC-PCR described by Versalovic et al. can efficiently analyze the variation of different ecological systems and monitor the development of microbial flora of the same ecological system. In recent years, this technology has been applied to study the microbial population of animal intestinal tract.

Abstract:Repeat sequence exists in almost all organism genomes. Enterobacterial Repetitive Intergenic Consensus (ERIC), also named Intergenic Repetitive Unit (IRU), is a kind of intergenic repetitive sequence that exists predominantly in Enterobacteria. ERIC(IRU) was firstly discovered in Escherichia coli, followed by identification in many other bacteria. ERIC(IRU) is 127bp long, and some with inserted sequences. Most ERIC(IRU) can be transcribed, and mRNA forms a stem-loop structure. ERIC(IRU) is restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames(ORF). ERIC(IRU) probably modulates the expression of flanking genes. ERIC(IRU) is highly conserved, for either its variation is restricted by the natural selection pressure, or it's “selfish DNA". ERIC-PCR described by Versalovic et al. can efficiently analyze the variation of different ecological systems and monitor the development of microbial flora of the same ecological system. In recent years, this technology has been applied to study the microbial population of animal intestinal tract.