CAO Hai-jun , LI Yong-wen , LEI Yu , WU Jiang , XU Heng
2007, 47(1):1-6.
Abstract:In October 2005, a large number of adults of Silurus meridionalis Chen died in the mud fish farming of Sichuan province. Later, three predominate strains of bacteria were isolated from the body of moribund fish. By artificial infection tests, strain TWN3 was confirmed to be the pathogen of the disease. Based on the characteristics of morphology, physiology and biochemistry tests, TWN3 was initially identified as Proteus vulgaris, and its G+C content of DNA is 39.1%. After being amplified, the sequence of its 16S rDNA was analyzed in the database of NCBI and it showed that TWN3 had the highest similarity to P. vulgaris, with 99.52% identity. By constructing the molecular phylogenetic dendrogram with Minimum Evolution method in Mega3.1, it was revealed that TWN3 was in the same branch with P. vulgaris. Based on all the results above, TWN3 is identified as P. vulgaris. However, the result of one biochemistry test, growth in KCN, deviates from the description in Bergey′s Manual of Systematic Bacteriology. With reference to the Manual above, Proteus vulgaris is divided into two groups, P. vulgaris BG2 and 3.According to the specific biochemical properties, TWN3 is classified as a member of P. vulgaris BG3. Relevant tests of biological properties were also conducted, which showed that this strain has no haemolysis and is sensitive to four kinds of antibiotic such as gentamicin. Moreover, it can strongly cause diseases to mice. The research on the growth property of strain TWN3 indicated that its growth temperature ranges from 10℃ to 43℃, optimum 37℃; growth pH ranges from 4 to 11, optimum 6.Its optimum salinity varies under different temperatures，and it grows best under 1.5% salinity while 37℃. The aim of these researches is to provide an evidence for the prevention and cure of TWN3. According to the appearance of the diseased Silurus meridionalis Chen and results of artificial infection test on crucian carps, it is considered that Silurus meridionalis Chen is infected through digestive system and it is also recommended that Bdellovibrio should be used in biological control. Although the pathogenicity of P. vulgaris is extensive, there has been no report that P. vulgaris is considered as the pathogen of cultivated Silurus meridionalis Chen so far. In addition，the identification of strain TWN3 has positive effects on the future research on the taxonomy of Proteus.
CAO Hai-jun , LI Yong-wen , LEI Yu , WU Jiang , XU Heng
2007, 47(1):1-6.
Abstract:In October 2005, a large number of adults of Silurus meridionalis Chen died in the mud fish farming of Sichuan province. Later, three predominate strains of bacteria were isolated from the body of moribund fish. By artificial infection tests, strain TWN3 was confirmed to be the pathogen of the disease. Based on the characteristics of morphology, physiology and biochemistry tests, TWN3 was initially identified as Proteus vulgaris, and its G+C content of DNA is 39.1%. After being amplified, the sequence of its 16S rDNA was analyzed in the database of NCBI and it showed that TWN3 had the highest similarity to P. vulgaris, with 99.52% identity. By constructing the molecular phylogenetic dendrogram with Minimum Evolution method in Mega3.1, it was revealed that TWN3 was in the same branch with P. vulgaris. Based on all the results above, TWN3 is identified as P. vulgaris. However, the result of one biochemistry test, growth in KCN, deviates from the description in Bergey′s Manual of Systematic Bacteriology. With reference to the Manual above, Proteus vulgaris is divided into two groups, P. vulgaris BG2 and 3.According to the specific biochemical properties, TWN3 is classified as a member of P. vulgaris BG3. Relevant tests of biological properties were also conducted, which showed that this strain has no haemolysis and is sensitive to four kinds of antibiotic such as gentamicin. Moreover, it can strongly cause diseases to mice. The research on the growth property of strain TWN3 indicated that its growth temperature ranges from 10℃ to 43℃, optimum 37℃; growth pH ranges from 4 to 11, optimum 6.Its optimum salinity varies under different temperatures，and it grows best under 1.5% salinity while 37℃. The aim of these researches is to provide an evidence for the prevention and cure of TWN3. According to the appearance of the diseased Silurus meridionalis Chen and results of artificial infection test on crucian carps, it is considered that Silurus meridionalis Chen is infected through digestive system and it is also recommended that Bdellovibrio should be used in biological control. Although the pathogenicity of P. vulgaris is extensive, there has been no report that P. vulgaris is considered as the pathogen of cultivated Silurus meridionalis Chen so far. In addition，the identification of strain TWN3 has positive effects on the future research on the taxonomy of Proteus.
ZHANG Lei , LIU Chun-qin , GAO Zhi-hui , CAO Yu , BAI Gang
2007, 47(1):7-10.
Abstract:Actinomycete strain ZG0429 with high production of streptavidin was isolated from soil samples. Based on the results of morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis, strain ZG0429 was placed within the genus Streptomyces and identified as the species Streptomyces lavendulae. The production reached 201.0mg/L under our fermentation conditions. With the effective system of ammonium_sulfate deposition and gel filtration chromatography, streptavidin was successfully purified from the culture supernatant with an overall recovery of 76.87% and purity of 97.03％. This study illustrates an effective and inexpensive method to produce the aimed protein with favorable yield, high purity and desirable activity, so it provides a valuable reference for the large_scale industrial production and isolation of streptavidin.
ZHANG Lei , LIU Chun-qin , GAO Zhi-hui , CAO Yu , BAI Gang
2007, 47(1):7-10.
Abstract:Actinomycete strain ZG0429 with high production of streptavidin was isolated from soil samples. Based on the results of morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis, strain ZG0429 was placed within the genus Streptomyces and identified as the species Streptomyces lavendulae. The production reached 201.0mg/L under our fermentation conditions. With the effective system of ammonium_sulfate deposition and gel filtration chromatography, streptavidin was successfully purified from the culture supernatant with an overall recovery of 76.87% and purity of 97.03％. This study illustrates an effective and inexpensive method to produce the aimed protein with favorable yield, high purity and desirable activity, so it provides a valuable reference for the large_scale industrial production and isolation of streptavidin.
SU Chun-li , TANG Chuan-hong , ZHANG Jing-song , CHEN Ming-jie , PAN Ying-jie
2007, 47(1):11-16.
Abstract:Nuclear ribosomal DNA ITS sequences have been used to investigate phylogenetic relationships between 34 Ganoderma isolates cultivated in China. Five distinct groups were identified: the subgenus Elfvingia, the sect. Phaeonema, and three groups within the sect. Ganoderma. Most of the Ganoderma isolates (85.7%) formed a single group within the sect. Ganoderma. The result indicated clear genetic diversity between the subgenus Elfvingia and the sects Phaeonema and Ganoderma, but a smaller degree of genetic diversity between the three groups placed within the sect. Ganoderma. Analysis of molecular data is a more effective and useful approach for studying the taxonomy of Ganoderma, and for stablishing phylogenetic relationships within the genus, compared to methods based on fruiting body morphology.
SU Chun-li , TANG Chuan-hong , ZHANG Jing-song , CHEN Ming-jie , PAN Ying-jie
2007, 47(1):11-16.
Abstract:Nuclear ribosomal DNA ITS sequences have been used to investigate phylogenetic relationships between 34 Ganoderma isolates cultivated in China. Five distinct groups were identified: the subgenus Elfvingia, the sect. Phaeonema, and three groups within the sect. Ganoderma. Most of the Ganoderma isolates (85.7%) formed a single group within the sect. Ganoderma. The result indicated clear genetic diversity between the subgenus Elfvingia and the sects Phaeonema and Ganoderma, but a smaller degree of genetic diversity between the three groups placed within the sect. Ganoderma. Analysis of molecular data is a more effective and useful approach for studying the taxonomy of Ganoderma, and for stablishing phylogenetic relationships within the genus, compared to methods based on fruiting body morphology.
YAN Xiao-hua , LIU Gang , TAN Hua-rong
2007, 47(1):17-21.
Abstract:In Bacilli, gerM is a very conservative gene. Primers were designed according to the gerM gene sequence of Bacillus cereus, and a 640bp DNA fragment was obtained from Bacillus thuringiensis subsp. kustaki 1.175 by PCR. Using this fragment as a probe, a 4.5kb DNA fragment was cloned from the partial DNA library of Bacillus thuringiensis subsp. kustaki 1.175.Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 349-amino acid (aa) protein, which has high homology with GerM protein from Bacillus subtilis. This gene was designated gerM (GenBank Accession No. DQ537381). RT-PCR analysis showed that gerM gene was only expressed in the process of sporulation, suggesting gerM is not required for the vegetative growth. The function of the gerM gene was studied by a strategy of gene disruption, and the resulting gerM disruption mutant did show normal growth and sporulation. However, gerM disruption mutant spores germinate slower than wild-type spores when triggered by L-alanine or inosine, indicating that gerM is required for the spore normal germination initiated by L-alanine or inosine in Bacillus thuringiensis.
YAN Xiao-hua , LIU Gang , TAN Hua-rong
2007, 47(1):17-21.
Abstract:In Bacilli, gerM is a very conservative gene. Primers were designed according to the gerM gene sequence of Bacillus cereus, and a 640bp DNA fragment was obtained from Bacillus thuringiensis subsp. kustaki 1.175 by PCR. Using this fragment as a probe, a 4.5kb DNA fragment was cloned from the partial DNA library of Bacillus thuringiensis subsp. kustaki 1.175.Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 349-amino acid (aa) protein, which has high homology with GerM protein from Bacillus subtilis. This gene was designated gerM (GenBank Accession No. DQ537381). RT-PCR analysis showed that gerM gene was only expressed in the process of sporulation, suggesting gerM is not required for the vegetative growth. The function of the gerM gene was studied by a strategy of gene disruption, and the resulting gerM disruption mutant did show normal growth and sporulation. However, gerM disruption mutant spores germinate slower than wild-type spores when triggered by L-alanine or inosine, indicating that gerM is required for the spore normal germination initiated by L-alanine or inosine in Bacillus thuringiensis.
LUO Qin , ZHOU Qing-chun , DENG Ling-fu , GAO Qiang , LIU De-li
2007, 47(1):22-28.
Abstract:To study some essential elements of a PrfA-dependent promoter of Listeria monocytogenes, a series of promoter mutants incorporated into upstream of a promoterless lacZ gene were constructed from a known listerial PrfA-dependent promoter, inlC promoter, by PCR-mediated site-directed mutagenesis and recombinant PCR technique and then electroporated into L. monocytogenes wild-type strain P14, prfA* mutant P14a and prfA deletion mutant A42.The corresponding transcription activities of altered promoters were measured by the β-galactosidase assay. The results showed that a PrfA-box-like sequence (“pseudo-PrfA-box”), TTAACAGCGTTTGTTAA, 22bp downstream of the transcriptional start site of PinlC had no ability to enhance or inhibit the PrfA-dependent transcription of inlC promoter, even it was modified to the “ideal PrfA-box” TTAACATTTGTTAA. However, there was almost no PrfA-dependent transcriptional activity from the mutants deletion of the inlC original PrfA-box. Moreover, altered spacing between 3′-end of the PrfA-box and 5′-end of the -10 box in the inlC promoter region affected transcription efficiency dramatically, which also happened in another promoter-dependent promoter, plcA promoter. Those above suggested that besides the “PrfA-box", additional unknown PrfA-dependent promoter structure(s) or sequence(s) might be required for the PrfA binding to the promoter and initiation of transcription. Furthermore, the distance between the PrfA-box and the -10 box should be fixed to 22 or 23bp for the PrfA-dependent transcription.
LUO Qin , ZHOU Qing-chun , DENG Ling-fu , GAO Qiang , LIU De-li
2007, 47(1):22-28.
Abstract:To study some essential elements of a PrfA-dependent promoter of Listeria monocytogenes, a series of promoter mutants incorporated into upstream of a promoterless lacZ gene were constructed from a known listerial PrfA-dependent promoter, inlC promoter, by PCR-mediated site-directed mutagenesis and recombinant PCR technique and then electroporated into L. monocytogenes wild-type strain P14, prfA* mutant P14a and prfA deletion mutant A42.The corresponding transcription activities of altered promoters were measured by the β-galactosidase assay. The results showed that a PrfA-box-like sequence (“pseudo-PrfA-box”), TTAACAGCGTTTGTTAA, 22bp downstream of the transcriptional start site of PinlC had no ability to enhance or inhibit the PrfA-dependent transcription of inlC promoter, even it was modified to the “ideal PrfA-box” TTAACATTTGTTAA. However, there was almost no PrfA-dependent transcriptional activity from the mutants deletion of the inlC original PrfA-box. Moreover, altered spacing between 3′-end of the PrfA-box and 5′-end of the -10 box in the inlC promoter region affected transcription efficiency dramatically, which also happened in another promoter-dependent promoter, plcA promoter. Those above suggested that besides the “PrfA-box", additional unknown PrfA-dependent promoter structure(s) or sequence(s) might be required for the PrfA binding to the promoter and initiation of transcription. Furthermore, the distance between the PrfA-box and the -10 box should be fixed to 22 or 23bp for the PrfA-dependent transcription.
CHENG Chang , CHEN Zhen , ZHU Cheng
2007, 47(1):29-33.
Abstract:Helicobacter pylori (Hp) is the principal cause of most chronic active gastric and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current vaccines are expensive to produce and deliver, however, transgenic plants expressing recombinant vaccine immunogens offer an attractive and potential inexpensive alternative to vaccination and injection. In this study, plant expression vectors which harbor Hp related proteins CagA and UreB were constructed. Fusion gene ctb-linker-cagA and ctb-linker-ureB were cut from vectors p1300-WxCLCN and p1300-WxCLUN, and then constructed into vector pCAMBIA2301 which was under the control of the CaMV 35S promoter by series molecular methods. Those reconstructed vectors were named p2301-35SCLCN and p2301-35SCLUN and were introduced into Agrobacterium tumefaciens strain EHA105.Tobacco was transformed by co-cultivating leaf discs with Agrobacterium strains harboring fusion genes. The regenerated Kanamycin-resistant transforms were selected, elongated, rooted and transferred for flowering in greenhouse. Recombinant plant expression vectors were confirmed by digestion and PCR and transgenic plants were analyzed by PCR, GUS histochemical assays, PCR-Southern blot. The results show that more than 80% transgenic plants are confirmed to be positive ones and these results also indicate that ctb-linker-cagA and ctb-linker-ureB are integrated into the genomic DNA of the tobacco which laid a solid foundation for the research of establishing transgenic plants as bioreactors to carry microbe antigen and Hp transgenic plant vaccines.
CHENG Chang , CHEN Zhen , ZHU Cheng
2007, 47(1):29-33.
Abstract:Helicobacter pylori (Hp) is the principal cause of most chronic active gastric and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current vaccines are expensive to produce and deliver, however, transgenic plants expressing recombinant vaccine immunogens offer an attractive and potential inexpensive alternative to vaccination and injection. In this study, plant expression vectors which harbor Hp related proteins CagA and UreB were constructed. Fusion gene ctb-linker-cagA and ctb-linker-ureB were cut from vectors p1300-WxCLCN and p1300-WxCLUN, and then constructed into vector pCAMBIA2301 which was under the control of the CaMV 35S promoter by series molecular methods. Those reconstructed vectors were named p2301-35SCLCN and p2301-35SCLUN and were introduced into Agrobacterium tumefaciens strain EHA105.Tobacco was transformed by co-cultivating leaf discs with Agrobacterium strains harboring fusion genes. The regenerated Kanamycin-resistant transforms were selected, elongated, rooted and transferred for flowering in greenhouse. Recombinant plant expression vectors were confirmed by digestion and PCR and transgenic plants were analyzed by PCR, GUS histochemical assays, PCR-Southern blot. The results show that more than 80% transgenic plants are confirmed to be positive ones and these results also indicate that ctb-linker-cagA and ctb-linker-ureB are integrated into the genomic DNA of the tobacco which laid a solid foundation for the research of establishing transgenic plants as bioreactors to carry microbe antigen and Hp transgenic plant vaccines.
TAO Wei-xin , WU Jing , DENG Zi-xin , TAO Mei-feng
2007, 47(1):34-38.
Abstract:bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in Streptomyces coelicolor. bldA inactivation leaded to defection in morphological development and production of two of four known antibiotics in S. coelicolor. A bldA homologue, termed bldAa, has been identified in the sequenced genome of Streptomyces avermitilis MA4680.To investigate the function of bldAa, genomic DNA of S. avermitilis NRRL8165 was digested with BamHⅠ and the 5～6kb was fractioned and ligated with the BamHⅠ digested E.coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldAa and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldAa replacement plasmid, was constructed using the λRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRL8165.Three bldA-disruption mutant strains (named TW10) were obtained, which showed a bald phenotype, indicating that bldAa controlled the morphological differentiation of S. avermitilis. HPLC analysis of the TW10 fermentation culture showed that TW10 did not synthesize avermectins anymore, suggesting that the synthesis of avermectins were dominated by bldAa. There are TTA codons within aveA3 and aveR of the avermectin biosynthesis gene cluster, suggesting that the translation of the two genes may depend on bldAa, which were consistent with the experimental results.
TAO Wei-xin , WU Jing , DENG Zi-xin , TAO Mei-feng
2007, 47(1):34-38.
Abstract:bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in Streptomyces coelicolor. bldA inactivation leaded to defection in morphological development and production of two of four known antibiotics in S. coelicolor. A bldA homologue, termed bldAa, has been identified in the sequenced genome of Streptomyces avermitilis MA4680.To investigate the function of bldAa, genomic DNA of S. avermitilis NRRL8165 was digested with BamHⅠ and the 5～6kb was fractioned and ligated with the BamHⅠ digested E.coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldAa and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldAa replacement plasmid, was constructed using the λRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRL8165.Three bldA-disruption mutant strains (named TW10) were obtained, which showed a bald phenotype, indicating that bldAa controlled the morphological differentiation of S. avermitilis. HPLC analysis of the TW10 fermentation culture showed that TW10 did not synthesize avermectins anymore, suggesting that the synthesis of avermectins were dominated by bldAa. There are TTA codons within aveA3 and aveR of the avermectin biosynthesis gene cluster, suggesting that the translation of the two genes may depend on bldAa, which were consistent with the experimental results.
2007, 47(1):39-43.
Abstract:RNA silencing is a highly conserved mechanism in many eukaryotic organisms that involves sequence-specific RNA degradation. One of the important roles of RNA silencing is antiviral infection. To counter this defense mechanism, many plant viruses have evolved or acquired functional proteins (suppressors) for suppression of RNA silencing. Geminiviruses are a family of small circular single-stranded DNA (ssDNA) viruses that cause severe diseases in major crop plants worldwide. AC2 (C2) and AC4 (C4) proteins of many geminiviruses were reported to be suppressors of RNA silencing. Ageratum yellow vein China virus (AYVCNV) and Stachytarpheta leaf curl virus are two distinct geminiviruses identified in China. In this study, the functions of C4 of AYVCNV and StaLCV were studied, analysis of infectivity of AYVCNV and StaLCV C4 genes using Tobacco rattle virus (TRV) vector were carried out in Nicotiana benthamiana plants, N. benthamiana plants expressing C4 can induce the virus-like symptoms, indicating that the C4 gene of AYVCNV or StaLCV is symptom determinant. In experiment of suppression of RNA silencing, C4 protein of AYVCNV or StaLCV can suppresses the local silencing of gfp in transgenic N. benthamiana plants (16c), suggests that both of them are suppressors of RNA silencing. The above results indicate that C4 of AYVCNV or StaLCV is a symptom determinant and suppressor of RNA silencing.
2007, 47(1):39-43.
Abstract:RNA silencing is a highly conserved mechanism in many eukaryotic organisms that involves sequence-specific RNA degradation. One of the important roles of RNA silencing is antiviral infection. To counter this defense mechanism, many plant viruses have evolved or acquired functional proteins (suppressors) for suppression of RNA silencing. Geminiviruses are a family of small circular single-stranded DNA (ssDNA) viruses that cause severe diseases in major crop plants worldwide. AC2 (C2) and AC4 (C4) proteins of many geminiviruses were reported to be suppressors of RNA silencing. Ageratum yellow vein China virus (AYVCNV) and Stachytarpheta leaf curl virus are two distinct geminiviruses identified in China. In this study, the functions of C4 of AYVCNV and StaLCV were studied, analysis of infectivity of AYVCNV and StaLCV C4 genes using Tobacco rattle virus (TRV) vector were carried out in Nicotiana benthamiana plants, N. benthamiana plants expressing C4 can induce the virus-like symptoms, indicating that the C4 gene of AYVCNV or StaLCV is symptom determinant. In experiment of suppression of RNA silencing, C4 protein of AYVCNV or StaLCV can suppresses the local silencing of gfp in transgenic N. benthamiana plants (16c), suggests that both of them are suppressors of RNA silencing. The above results indicate that C4 of AYVCNV or StaLCV is a symptom determinant and suppressor of RNA silencing.
WANG Dong-liang , XIAO Min , QIAN Wei , HAN Bo
2007, 47(1):44-47.
Abstract:Selenium is essential element for humans and animals but is very toxic at higher concentrations. In four inorganic states of selenate ［SeO2-４(Ⅵ)］, selenite ［SeO2-３(Ⅳ)］, elemental selenium ［Se°(0)］and selenide ［Se2-(-Ⅱ)］, selenite is well known to be more soluble and higher toxic than other three forms. Many microorganisms have the capacity to reduce selenite to red elemental selenium, which provide the potential to cope with the detoxification of pollution and to use the biological availability of red elemental selenium. Strain S3 that was more resistant to sodium selenite was selected from 20 photosynthetic bacteria preserved in laboratory. The red granule produced by S3 was identified as elemental selenium (Se°) by transmission electron microscopy and Electron-Dispersive X-ray (EDX) analysis. The granule diameter of the red elemental selenium was 5nm～200nm, similar as the Nano-Se that has bioavailability. Morphology, physiology and photosynthetic pigments analysis results showed that strain S3 was essentially consistent with Rhodobacter azotoformans. The 16S rDNA sequence analysis (GenBank accession number DQ402051) suggested that strain S3 was clustered together with R. azotoformans in phylogenetic tree, with the sequence identity of 99%. Based on all the results of taxonomy, strain S3 was identified as R. azotoformans S3.The effects of selenite on growth kinetics and the ability to resistant selenite of strain S3 were investigated. In contrast to Rhodospirillum rubrum which was reported not to reduce selenite until the end of exponential growth, strain S3 transformed selenite (1.25mmol/L) at the beginning of the growth, suggesting that strain S3 and Rs. rubrum may employ different strategies to reduce selenite. Strain S3 can grow in the presence of up to 125mmol/L sodium selenite, which is much higher than those which could be resisted to by other bacteria such as Escherichia coli (<20mmol/L) and Ralstonia metallidurans CH34 (<6mmol/L). It is firstly reported that R. azotoformans has the capacity to reduce selenite to red elemental selenium.
WANG Dong-liang , XIAO Min , QIAN Wei , HAN Bo
2007, 47(1):44-47.
Abstract:Selenium is essential element for humans and animals but is very toxic at higher concentrations. In four inorganic states of selenate ［SeO2-４(Ⅵ)］, selenite ［SeO2-３(Ⅳ)］, elemental selenium ［Se°(0)］and selenide ［Se2-(-Ⅱ)］, selenite is well known to be more soluble and higher toxic than other three forms. Many microorganisms have the capacity to reduce selenite to red elemental selenium, which provide the potential to cope with the detoxification of pollution and to use the biological availability of red elemental selenium. Strain S3 that was more resistant to sodium selenite was selected from 20 photosynthetic bacteria preserved in laboratory. The red granule produced by S3 was identified as elemental selenium (Se°) by transmission electron microscopy and Electron-Dispersive X-ray (EDX) analysis. The granule diameter of the red elemental selenium was 5nm～200nm, similar as the Nano-Se that has bioavailability. Morphology, physiology and photosynthetic pigments analysis results showed that strain S3 was essentially consistent with Rhodobacter azotoformans. The 16S rDNA sequence analysis (GenBank accession number DQ402051) suggested that strain S3 was clustered together with R. azotoformans in phylogenetic tree, with the sequence identity of 99%. Based on all the results of taxonomy, strain S3 was identified as R. azotoformans S3.The effects of selenite on growth kinetics and the ability to resistant selenite of strain S3 were investigated. In contrast to Rhodospirillum rubrum which was reported not to reduce selenite until the end of exponential growth, strain S3 transformed selenite (1.25mmol/L) at the beginning of the growth, suggesting that strain S3 and Rs. rubrum may employ different strategies to reduce selenite. Strain S3 can grow in the presence of up to 125mmol/L sodium selenite, which is much higher than those which could be resisted to by other bacteria such as Escherichia coli (<20mmol/L) and Ralstonia metallidurans CH34 (<6mmol/L). It is firstly reported that R. azotoformans has the capacity to reduce selenite to red elemental selenium.
CHEN Xiao-fang , ZHAO Zhi , ZHANG Ying-zi , WANG Yu , DING Jiu-yuan
2007, 47(1):48-53.
Abstract:Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67.The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.
CHEN Xiao-fang , ZHAO Zhi , ZHANG Ying-zi , WANG Yu , DING Jiu-yuan
2007, 47(1):48-53.
Abstract:Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67.The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.
LI Jian-feng , HONG Yu-zhi , XIAO Ya-zhong
2007, 47(1):54-58.
Abstract:A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5′-and 3′-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20℃ in BMM medium containing 0.3mmol/L CuSO4 and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62×104 U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.
LI Jian-feng , HONG Yu-zhi , XIAO Ya-zhong
2007, 47(1):54-58.
Abstract:A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5′-and 3′-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20℃ in BMM medium containing 0.3mmol/L CuSO4 and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62×104 U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.
LU He , CHAI You-rong , ZHANG Xue-kun , LEI Tian-gang , LI Jia-na
2007, 47(1):59-63.
Abstract:Fatty acid composition of fungi is analysed through the gas chromatography(GC) technique. With specific activity a novel enzyme △6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R.stolonifer was identified as a fungal species that produced plentiful γ-linolenic acid. A 1475bp full-length cDNA, designated as RnD6Dhere, with high homology to fungal △6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of his novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to γ-linolenic acid(GLA) methyl ester standards was detected with the same retention time,which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced △6-fatty acid desaturase activity of RnD6D which led to the accumulation of γ-linolenic acid.
LU He , CHAI You-rong , ZHANG Xue-kun , LEI Tian-gang , LI Jia-na
2007, 47(1):59-63.
Abstract:Fatty acid composition of fungi is analysed through the gas chromatography(GC) technique. With specific activity a novel enzyme △6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R.stolonifer was identified as a fungal species that produced plentiful γ-linolenic acid. A 1475bp full-length cDNA, designated as RnD6Dhere, with high homology to fungal △6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of his novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to γ-linolenic acid(GLA) methyl ester standards was detected with the same retention time,which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced △6-fatty acid desaturase activity of RnD6D which led to the accumulation of γ-linolenic acid.
HUANG Zhi-hua , ZHANG Yan-ping , LIU Ming , WANG Bao-guang , CAO Zhu-an
2007, 47(1):64-68.
Abstract:Glycerol can be converted to 1,3-propanediol by the anaerobic fermentation of Klebsiella pneumoniae, during which reducing equivalent NADH was consumed. Therefore, the availability of NADH would be critical for the yield of 1,3-propanediol. In this paper, formate/formate dehydrogenase system was used for the regeneration of in vivo NADH and the improvement of 1,3-propanediol production. Formate Dehydrogenase gene (fdh) was amplified from Candida boidinii genome by PCR and the purified PCR product was inserted into the vector pMD18-T Simple to construct plasmid pMD18-T Simple-fdh, which was transformed into Escherichia coli DH5α and recombinants were selected by blue-white selection. From the transformant the fdh gene was separated and inserted into pMALTM-p2X to construct expression vector pMALTM-p2X-fdh, which was transformed into Klebsiella pneumoniae YMU2 and a recombinant strain Klebsiella pneumoniae F-1 was obtained. The plasmid stability of strain F-1 and the conditions of fdh expression induced by IPTG were studied. It was demonstrated that the plasmid had good stability, and 0.5mmol/L IPTG would induce the expression of protein encoded by fdh gene with the molecular weight of 40.2kDa. The enzyme activity reached 5.47U/mg crude protein when K. pneumoniae F-1 was induced for 4h by 0.5mmol/L IPTG. Compared with that of the parent strain K. pneumoniae YMU2, the yield of 1,3-propanediol of recombinant strain F-1 increased by 12.5% in the anaeribic bioreactor.
HUANG Zhi-hua , ZHANG Yan-ping , LIU Ming , WANG Bao-guang , CAO Zhu-an
2007, 47(1):64-68.
Abstract:Glycerol can be converted to 1,3-propanediol by the anaerobic fermentation of Klebsiella pneumoniae, during which reducing equivalent NADH was consumed. Therefore, the availability of NADH would be critical for the yield of 1,3-propanediol. In this paper, formate/formate dehydrogenase system was used for the regeneration of in vivo NADH and the improvement of 1,3-propanediol production. Formate Dehydrogenase gene (fdh) was amplified from Candida boidinii genome by PCR and the purified PCR product was inserted into the vector pMD18-T Simple to construct plasmid pMD18-T Simple-fdh, which was transformed into Escherichia coli DH5α and recombinants were selected by blue-white selection. From the transformant the fdh gene was separated and inserted into pMALTM-p2X to construct expression vector pMALTM-p2X-fdh, which was transformed into Klebsiella pneumoniae YMU2 and a recombinant strain Klebsiella pneumoniae F-1 was obtained. The plasmid stability of strain F-1 and the conditions of fdh expression induced by IPTG were studied. It was demonstrated that the plasmid had good stability, and 0.5mmol/L IPTG would induce the expression of protein encoded by fdh gene with the molecular weight of 40.2kDa. The enzyme activity reached 5.47U/mg crude protein when K. pneumoniae F-1 was induced for 4h by 0.5mmol/L IPTG. Compared with that of the parent strain K. pneumoniae YMU2, the yield of 1,3-propanediol of recombinant strain F-1 increased by 12.5% in the anaeribic bioreactor.
LIU Gang , ZHANG Yan , LI Yun , YU Shao-wen , XING Miao
2007, 47(1):69-74.
Abstract:Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.
LIU Gang , ZHANG Yan , LI Yun , YU Shao-wen , XING Miao
2007, 47(1):69-74.
Abstract:Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.
WANG Xiu-qing , SU Chun-xia , ZHOU-Bin , CAO Rui-bing , CHEN Pu-yan
2007, 47(1):75-78.
Abstract:According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZα-A to construct the recombinant expression vector pPICZα-A-CA. The SacI-linearized plasmid pPICZα-A-CA was transformed into P. pastoris SMD1168 by electroporation. Under the control of the promoter AOX(alcoholoxidase), an approximately 1.9kDa cecA-mag protein was expressed. Antibacterial assays demonstrated that cecA-mag had broad spectrum of antimicrobial property against Gram-positive as well as Gram-negative bacteria especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E. coli. In addition, the hybrid antibacterial peptide showed an extreme heat stable and acid stable characteristic. These results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional cecA-mag for both research and industrial purpose.Based on these characteristics, the recombinant antibacterial peptide cecA-mag displays application foreground in the field of prevention of disease, and can be used as additives of animal feedstuff and so on.
WANG Xiu-qing , SU Chun-xia , ZHOU-Bin , CAO Rui-bing , CHEN Pu-yan
2007, 47(1):75-78.
Abstract:According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZα-A to construct the recombinant expression vector pPICZα-A-CA. The SacI-linearized plasmid pPICZα-A-CA was transformed into P. pastoris SMD1168 by electroporation. Under the control of the promoter AOX(alcoholoxidase), an approximately 1.9kDa cecA-mag protein was expressed. Antibacterial assays demonstrated that cecA-mag had broad spectrum of antimicrobial property against Gram-positive as well as Gram-negative bacteria especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E. coli. In addition, the hybrid antibacterial peptide showed an extreme heat stable and acid stable characteristic. These results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional cecA-mag for both research and industrial purpose.Based on these characteristics, the recombinant antibacterial peptide cecA-mag displays application foreground in the field of prevention of disease, and can be used as additives of animal feedstuff and so on.
HOU Xin , HU Ting-mao , LIU Jun-e
2007, 47(1):79-82.
Abstract:STK11(serine/threonine kinase 11), a multi-functional protein reported recently, possibly participates in a broad range of cellular processes, including regulation of cell cycle, p53-mediated apoptosis, ras-induced cell transformation and cell polarization. An efficient expression of functional STK11 in Escherichia coli will promote the study on its structure and function. Inducible prokaryotic expression vector pET-Nus-STK11(with Nus fusion tag) was constructed with pET-44a(+) and the cDNA of STK11 gene cloned in our lab. pET-Nus-STK11 was then expressed in both BL21(DE3) and Rosetta-gami (DE3)pLysS on the induction of IPTG. SDS-PAGE and Western blot indicated that recombinant Nus-STK11 obtained in BL21(DE3) was in the form of inclusion body, whereas that from Rosetta-gami (DE3)pLysS was mainly in soluble fraction, and accounted for 8.9% and 16.7% of the total protein, respectively. After purification and refolding, the obtained recombinant protein was carried into SMMC-7721 cells by Chariot to observe its influence on cell growth and cell cycle. Nus-STK11 from BL21(DE3) was proved to be lack of any tumor-suppression activity, while a growth inhibitory ratio of 47.05% on SMMC-7721 cell was observed, and cell cycle progression of SMMC-7721 cells was also arrested from G0/G1 to S phase, with the Nus-STK11 from Rosetta-gami (DE3)pLysS, indicating that the above recombinant fusion protein from Rosetta-gami (DE3)pLysS had significant biological activity. This is the first report on functional recombinant STK11 protein expressed in Escherichia coli.
HOU Xin , HU Ting-mao , LIU Jun-e
2007, 47(1):79-82.
Abstract:STK11(serine/threonine kinase 11), a multi-functional protein reported recently, possibly participates in a broad range of cellular processes, including regulation of cell cycle, p53-mediated apoptosis, ras-induced cell transformation and cell polarization. An efficient expression of functional STK11 in Escherichia coli will promote the study on its structure and function. Inducible prokaryotic expression vector pET-Nus-STK11(with Nus fusion tag) was constructed with pET-44a(+) and the cDNA of STK11 gene cloned in our lab. pET-Nus-STK11 was then expressed in both BL21(DE3) and Rosetta-gami (DE3)pLysS on the induction of IPTG. SDS-PAGE and Western blot indicated that recombinant Nus-STK11 obtained in BL21(DE3) was in the form of inclusion body, whereas that from Rosetta-gami (DE3)pLysS was mainly in soluble fraction, and accounted for 8.9% and 16.7% of the total protein, respectively. After purification and refolding, the obtained recombinant protein was carried into SMMC-7721 cells by Chariot to observe its influence on cell growth and cell cycle. Nus-STK11 from BL21(DE3) was proved to be lack of any tumor-suppression activity, while a growth inhibitory ratio of 47.05% on SMMC-7721 cell was observed, and cell cycle progression of SMMC-7721 cells was also arrested from G0/G1 to S phase, with the Nus-STK11 from Rosetta-gami (DE3)pLysS, indicating that the above recombinant fusion protein from Rosetta-gami (DE3)pLysS had significant biological activity. This is the first report on functional recombinant STK11 protein expressed in Escherichia coli.
ZHANG Tao , LIU Shuang-jiang , LIU Zhi-pei
2007, 47(1):83-87.
Abstract:Up to date, p-chloroaniline was reported to be biodegraded only through ortho-or modified ortho-cleavage pathway. In this paper, the intermediates of p-chloroaniline metabolized by strain Diaphorobacter PCA039 were analyzed and identified by HPLC and LC/MS methods. The results showed that intermediates such as 4-chlorocatechol, 2-hydroxy-5-chloromuconic semialdehyde, 5-chloro-4-oxalocrotonate, 5-chloro-2-oxo-4-hydroxypentanote, chloro-acetate were formed and consumed in order during the degradation of p-chloroaniline by strain Diaphorobacter PCA039.These compounds are the intermediates of a typical meta-cleavage pathway. In addition, strain Diaphorobacter PCA039 could produce the induced catechol 2, 3-dioxygenase when it grows on p-chloroaniline as sole carbon, nitrogen and energy sources, and use the intermediates of the meta-cleavage pathway such as catechol, 4-chlorocatechol, 2-hydroxy-5-chloromuconic semialdehyde as sole carbon and energy sources. Combining its physiological and biochemical properties and the results of LC/MS analysis, it was concluded that strain Diaphorobacter PCA039 could also degrade p-chloroaniline through meta-cleavage pathway as Fig. 4, which is a new pathway for the biodegradation of p-chloroaniline. It is significant to the study of biodegradation, metabolic mechanism, properties of the enzymes and the expression and regulation of the genes involved in the degradation of p-chloroaniline.
ZHANG Tao , LIU Shuang-jiang , LIU Zhi-pei
2007, 47(1):83-87.
Abstract:Up to date, p-chloroaniline was reported to be biodegraded only through ortho-or modified ortho-cleavage pathway. In this paper, the intermediates of p-chloroaniline metabolized by strain Diaphorobacter PCA039 were analyzed and identified by HPLC and LC/MS methods. The results showed that intermediates such as 4-chlorocatechol, 2-hydroxy-5-chloromuconic semialdehyde, 5-chloro-4-oxalocrotonate, 5-chloro-2-oxo-4-hydroxypentanote, chloro-acetate were formed and consumed in order during the degradation of p-chloroaniline by strain Diaphorobacter PCA039.These compounds are the intermediates of a typical meta-cleavage pathway. In addition, strain Diaphorobacter PCA039 could produce the induced catechol 2, 3-dioxygenase when it grows on p-chloroaniline as sole carbon, nitrogen and energy sources, and use the intermediates of the meta-cleavage pathway such as catechol, 4-chlorocatechol, 2-hydroxy-5-chloromuconic semialdehyde as sole carbon and energy sources. Combining its physiological and biochemical properties and the results of LC/MS analysis, it was concluded that strain Diaphorobacter PCA039 could also degrade p-chloroaniline through meta-cleavage pathway as Fig. 4, which is a new pathway for the biodegradation of p-chloroaniline. It is significant to the study of biodegradation, metabolic mechanism, properties of the enzymes and the expression and regulation of the genes involved in the degradation of p-chloroaniline.
JV Shou-yong , JI Fang , ZHU Zi-min , YU Zi-niu , SUN Ming
2007, 47(1):88-91.
Abstract:Parasporal crystal in B. thuringiensis subsp. finitimus T02 forms inside the exosporium and remains attached to the spore after mother cell lysis. Two crystal protein gene cry26Aa and cry28Aa were cloned from this strain. The relationship between the phenomenon of spore-crystal connection and the plasmids was investigated in this work. Mutants BMB1151 curing of the plasmid harboring cry26Aa, and BMB1152 curing of all plasmids were obtained from B. thuringiensis subsp. finitimus T02.The spore-crystal connection didn't appeared when cry26Aa and cry28Aa were transformed back into the strain BMB1152 by shuffle vector alone or in combination together. It suggested the plasmids of B. thuringiensis subsp. finitimus T02 may contribute to the phenomenon. When gene cry26Aa was transformed back into in the strain BMB1151, spore-crystal connection didn't form either. So the plasmid harboring cry26Aa may also contribute to the phenomenon of spore-crystal connection in B. thuringiensis subsp. finitimus T02.
JV Shou-yong , JI Fang , ZHU Zi-min , YU Zi-niu , SUN Ming
2007, 47(1):88-91.
Abstract:Parasporal crystal in B. thuringiensis subsp. finitimus T02 forms inside the exosporium and remains attached to the spore after mother cell lysis. Two crystal protein gene cry26Aa and cry28Aa were cloned from this strain. The relationship between the phenomenon of spore-crystal connection and the plasmids was investigated in this work. Mutants BMB1151 curing of the plasmid harboring cry26Aa, and BMB1152 curing of all plasmids were obtained from B. thuringiensis subsp. finitimus T02.The spore-crystal connection didn't appeared when cry26Aa and cry28Aa were transformed back into the strain BMB1152 by shuffle vector alone or in combination together. It suggested the plasmids of B. thuringiensis subsp. finitimus T02 may contribute to the phenomenon. When gene cry26Aa was transformed back into in the strain BMB1151, spore-crystal connection didn't form either. So the plasmid harboring cry26Aa may also contribute to the phenomenon of spore-crystal connection in B. thuringiensis subsp. finitimus T02.
LIAO Wei , SUN Fan , SONG Shao-yun , SHI Wei , PANG Yi
2007, 47(1):92-97.
Abstract:A Bacillus thuringiensis (Bt) fermentative strain MZ1 (subsp.kurstaki), from a company in Meixian County of Guangdong Province, produce toxins during sporulation and are extensively used in the field to control pest insects (Lepidoptera) in China. But some unknown or random factors that inhibited or stopped B.t growth in the fermentation can be regarded as reflecting the exist of lysogenic phage. Therefore, strain MZ1 and its lysogenic phages were studied in this paper. With indicator strain ZK1, two kind of phage plaques, one with about 3mm diameter and the other with about 1mm diameter, can be observed after strain MZ1 cultured in plates or flasks was induced by mitomycin C. Then, two lysogenic phages, namely MZTP01 and MZTP02, were isolated and characterized in biology. They belonged to family Siphoviridae, which had icosahedral heads (MZTP01:82nm×85nm; MZTP02: 75nm×55nm) and long tails (MZTP01: 220nm×18nm; MZTP02:183nm×12nm) without flexibility. Host range examination showed that six and seven (including indicator strain ZK1) out of 113 B.t strains saved in our laboratory were sensitive to MZTP01 and MZTP02, respectively. MZTP01 was more stable than MZTP02 against pH value, ultraviolet and heat treatment, but contrary against organic solvents. For MZTP01 and MZTP02, K values in the neutralization reactions were 45 and 326, respectively. Both phages had no relationship in their antigenicity. Burst size of phage MZTP02 was 175, two times more than that of MZTP01. Latent time of MZTP02 was 40min, one times shorter than that of MZTP01. It was suggested that both phages DNA be linear dsDNA through the typical absorption curves, reaction with diphenylamine, DNase sensitivity and acridine orange staining. And this was in good accord with the previous findings that all tailed phages being dsDNA moleculars. The genomic DNA and their restriction maps showed that both molecular weights should be between 9.4～23kb. Both phage genomic DNAs were digested by HindⅢ, producing eight and nine bands, respectively. The results above show that strain MZ1 is proved a double lysogen. It is considered to be the main reason of great losses in the production. The biological information of the two lysogenic phages belonging to strain MZ1 is provided to help solve the problem and the two lysogenic phages is prepared for complete DNA sequencing in the next step.
LIAO Wei , SUN Fan , SONG Shao-yun , SHI Wei , PANG Yi
2007, 47(1):92-97.
Abstract:A Bacillus thuringiensis (Bt) fermentative strain MZ1 (subsp.kurstaki), from a company in Meixian County of Guangdong Province, produce toxins during sporulation and are extensively used in the field to control pest insects (Lepidoptera) in China. But some unknown or random factors that inhibited or stopped B.t growth in the fermentation can be regarded as reflecting the exist of lysogenic phage. Therefore, strain MZ1 and its lysogenic phages were studied in this paper. With indicator strain ZK1, two kind of phage plaques, one with about 3mm diameter and the other with about 1mm diameter, can be observed after strain MZ1 cultured in plates or flasks was induced by mitomycin C. Then, two lysogenic phages, namely MZTP01 and MZTP02, were isolated and characterized in biology. They belonged to family Siphoviridae, which had icosahedral heads (MZTP01:82nm×85nm; MZTP02: 75nm×55nm) and long tails (MZTP01: 220nm×18nm; MZTP02:183nm×12nm) without flexibility. Host range examination showed that six and seven (including indicator strain ZK1) out of 113 B.t strains saved in our laboratory were sensitive to MZTP01 and MZTP02, respectively. MZTP01 was more stable than MZTP02 against pH value, ultraviolet and heat treatment, but contrary against organic solvents. For MZTP01 and MZTP02, K values in the neutralization reactions were 45 and 326, respectively. Both phages had no relationship in their antigenicity. Burst size of phage MZTP02 was 175, two times more than that of MZTP01. Latent time of MZTP02 was 40min, one times shorter than that of MZTP01. It was suggested that both phages DNA be linear dsDNA through the typical absorption curves, reaction with diphenylamine, DNase sensitivity and acridine orange staining. And this was in good accord with the previous findings that all tailed phages being dsDNA moleculars. The genomic DNA and their restriction maps showed that both molecular weights should be between 9.4～23kb. Both phage genomic DNAs were digested by HindⅢ, producing eight and nine bands, respectively. The results above show that strain MZ1 is proved a double lysogen. It is considered to be the main reason of great losses in the production. The biological information of the two lysogenic phages belonging to strain MZ1 is provided to help solve the problem and the two lysogenic phages is prepared for complete DNA sequencing in the next step.
YU Hong-yan , ZENG Guang-ming , XI Xing-mei , HUANG Hong-li , HUANG Guo-he
2007, 47(1):99-102.
Abstract:Bacterial communities were analyzed using Biolog method in agricultural waste composting. The results of cluster analysis and principle component analysis indicated that bacterial communities varied greatly during the first stage of composting, while began to stabilize during the second stage. Bacteria that could utilize the first and second kinds of carbon sources on Biolog plate were found to be the dominant ones during composting, which were also believed to be related with lignocellulose degradation. The bacteria utilizing the sixth kinds of carbon sources were just able to metabolize some simpler organic matters.
YU Hong-yan , ZENG Guang-ming , XI Xing-mei , HUANG Hong-li , HUANG Guo-he
2007, 47(1):99-102.
Abstract:Bacterial communities were analyzed using Biolog method in agricultural waste composting. The results of cluster analysis and principle component analysis indicated that bacterial communities varied greatly during the first stage of composting, while began to stabilize during the second stage. Bacteria that could utilize the first and second kinds of carbon sources on Biolog plate were found to be the dominant ones during composting, which were also believed to be related with lignocellulose degradation. The bacteria utilizing the sixth kinds of carbon sources were just able to metabolize some simpler organic matters.
LUO Ming-fang , WU Hao , WANG Lei , XING Xin-hui
2007, 47(1):103-109.
Abstract:From an agricultural sample taken in Chongqing, a stable methane-oxidizing mixed microbial consortium was established by enrichment culture with methane as a sole source of carbon and energy. The mixed consortium showed high capability of phenol degradation and 1,2-epoxypropane production from propene. More than 99% of phenol at an initial concentration of 600mg/L could be degraded by the mixed microbial consortium after 11 h of cultivation. The productivity of 1,2-epoxypropane could be increased with the decrease of phosphate concentration. The concentration of 1,2-epoxypropane produced could reach to 5.0mmol/L. The bacterial structure of the methane-oxidizing mixed microbial consortium was analyzed by pure culture isolation combining with 16S rRNA and PCR of the related MMO functional genes. The results showed that the methane-oxidizing mixed microbial consortium was composed of a type Ⅱ methanotroph identified as Methylosinus trichosporium and at least 4 kinds of heterotrophs (Comamonas testosteroni，Cupriavidus metallidurans, Acinetobacter junii and Stenotrophomonas maltophilia). M. trichosporium Y9, isolated from the mixed consortium, harbored both sMMO and pMMO genes.
LUO Ming-fang , WU Hao , WANG Lei , XING Xin-hui
2007, 47(1):103-109.
Abstract:From an agricultural sample taken in Chongqing, a stable methane-oxidizing mixed microbial consortium was established by enrichment culture with methane as a sole source of carbon and energy. The mixed consortium showed high capability of phenol degradation and 1,2-epoxypropane production from propene. More than 99% of phenol at an initial concentration of 600mg/L could be degraded by the mixed microbial consortium after 11 h of cultivation. The productivity of 1,2-epoxypropane could be increased with the decrease of phosphate concentration. The concentration of 1,2-epoxypropane produced could reach to 5.0mmol/L. The bacterial structure of the methane-oxidizing mixed microbial consortium was analyzed by pure culture isolation combining with 16S rRNA and PCR of the related MMO functional genes. The results showed that the methane-oxidizing mixed microbial consortium was composed of a type Ⅱ methanotroph identified as Methylosinus trichosporium and at least 4 kinds of heterotrophs (Comamonas testosteroni，Cupriavidus metallidurans, Acinetobacter junii and Stenotrophomonas maltophilia). M. trichosporium Y9, isolated from the mixed consortium, harbored both sMMO and pMMO genes.
LIU Ying , HONG Kui , ZHUANG Ling , LIN Hai-peng
2007, 47(1):110-114.
Abstract:Microbial natural products have been mainly acquired by pure culture. While in nature, different microorganisms in one habitat may work together as a whole to produce special secondary metabolites to adapt to their environments. “Quorum-sensing" is a way that they would use. A microbial consortium is like a multi-cellular individual that different microorganisms interact with each other to fulfill a special function. A marine fungus only produce antibiotics when a bacterium co-cultured (Cueto et al, 2001); and some traditional Chinese fermentation food are produced by mixed culture. These inspired us that directly using natural microbial consortium instead of isolate the individual microorganism may be a worth to risk in search for bioactive products. In this research, One hundred and eighty one samples were collected from three mangrove areas of Hainan, Guangxi and Guangdong, in China, which were fermented directly and evaluated for their anti-bacteria, anti-fungi and anti-cancer cell activities. Fifteen samples with high activities were further studied. Microorganisms were isolated from these 15 high bioactive samples and re-detected for their bioactivity, among which, microorganisms isolated from 5 samples that numbered 1106, 1122, 1116, 1214 and 1305 didn't show any activity although the un-isolated samples themselves showed high bioactivity. Four strains were isolated from one sample of number 1146. Among these strains, one strain showed the same bioactivity targets as the sample itself , but lower activity of anti-fungi than the sample itself. The other three strains didn't show any bioactivity. Microorganisms isolated from three samples that numbered 1161, 1123 and 1111, changed their initial bioactivity targets. These results suggested that natural microbial consortium culture have the potential to produce bioactive metabolites. It is supposed that some uncultured microorganisms or the community action may be the reason for their activity. This is an initial step on using microbial consortium to produce bioactive metabolites.
LIU Ying , HONG Kui , ZHUANG Ling , LIN Hai-peng
2007, 47(1):110-114.
Abstract:Microbial natural products have been mainly acquired by pure culture. While in nature, different microorganisms in one habitat may work together as a whole to produce special secondary metabolites to adapt to their environments. “Quorum-sensing" is a way that they would use. A microbial consortium is like a multi-cellular individual that different microorganisms interact with each other to fulfill a special function. A marine fungus only produce antibiotics when a bacterium co-cultured (Cueto et al, 2001); and some traditional Chinese fermentation food are produced by mixed culture. These inspired us that directly using natural microbial consortium instead of isolate the individual microorganism may be a worth to risk in search for bioactive products. In this research, One hundred and eighty one samples were collected from three mangrove areas of Hainan, Guangxi and Guangdong, in China, which were fermented directly and evaluated for their anti-bacteria, anti-fungi and anti-cancer cell activities. Fifteen samples with high activities were further studied. Microorganisms were isolated from these 15 high bioactive samples and re-detected for their bioactivity, among which, microorganisms isolated from 5 samples that numbered 1106, 1122, 1116, 1214 and 1305 didn't show any activity although the un-isolated samples themselves showed high bioactivity. Four strains were isolated from one sample of number 1146. Among these strains, one strain showed the same bioactivity targets as the sample itself , but lower activity of anti-fungi than the sample itself. The other three strains didn't show any bioactivity. Microorganisms isolated from three samples that numbered 1161, 1123 and 1111, changed their initial bioactivity targets. These results suggested that natural microbial consortium culture have the potential to produce bioactive metabolites. It is supposed that some uncultured microorganisms or the community action may be the reason for their activity. This is an initial step on using microbial consortium to produce bioactive metabolites.
2007, 47(1):115-120.
Abstract:To enhance the antibacterial ability of Magainin1-12，its N side was joined with an alkaline peptide named Hexapeptide(RRWQWR)，which would make Magainin1-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris，a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin(1-12). Synthesized through gene splicing by overlap extension,the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation，the hybrid antibacterial peptide was expressed on high level. The new peptide，which has a weight of 2.3kDa，could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay，Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.
2007, 47(1):115-120.
Abstract:To enhance the antibacterial ability of Magainin1-12，its N side was joined with an alkaline peptide named Hexapeptide(RRWQWR)，which would make Magainin1-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris，a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin(1-12). Synthesized through gene splicing by overlap extension,the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation，the hybrid antibacterial peptide was expressed on high level. The new peptide，which has a weight of 2.3kDa，could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay，Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.
WANG Yong-shan , FAN Hong-jie , ZHANG Xiao-min , LI Yin , XIAO Huan-juan
2007, 47(1):121-125.
Abstract:Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS.The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400μg per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1∶4000 at the 7th day after first immunization and 1∶256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (50μg per injection) and the resultant antibody titer was up to 1∶12800 at the 7th day after the second immunonization. After challenge with 200ELD50 of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.
WANG Yong-shan , FAN Hong-jie , ZHANG Xiao-min , LI Yin , XIAO Huan-juan
2007, 47(1):121-125.
Abstract:Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS.The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400μg per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1∶4000 at the 7th day after first immunization and 1∶256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (50μg per injection) and the resultant antibody titer was up to 1∶12800 at the 7th day after the second immunonization. After challenge with 200ELD50 of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.
JIN Xi-xin , CUI Bao-an , WEI Zhan-yong , LIU Zhan-tong , WANG Xue-bin
2007, 47(1):126-130.
Abstract:The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1.The insert position，the size and the frame were identified by PCR，restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37℃ induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body.Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.
JIN Xi-xin , CUI Bao-an , WEI Zhan-yong , LIU Zhan-tong , WANG Xue-bin
2007, 47(1):126-130.
Abstract:The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1.The insert position，the size and the frame were identified by PCR，restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37℃ induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body.Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.
JIAO Hong-mei , JIAO Xin-an , YIN Yue-lan , PAN Zhi-ming , MENG Song-shu
2007, 47(1):131-135.
Abstract:The complete S1 gene from mouse hepatitis virus (MHV) was amplified by RT-PCR and cloned into the pMD18-T vector. After confirmed by the restriction endonuclease analysis and PCR amplification, the positive clone of S1 gene was sequenced and then was transferred into eukaryotic expressing vector pVAX1. The recombinant plasmid pVAX1-S1 was transfected into COS-7 cells. The expressed S1 protein was successfully detected with indirect immunofluorescent assay. Finally, The recombinant plasmid pVAX1-S1 was transformed by electroporation into attenuated Salmonella typhimurium strain SL7207 and confirmed by PCR and Salmonella agglutination test. The recombinant was named as SL7207(pVAX1-S1). 6-week-old BALB/c mice were inoculated orally with SL7207 (pVAX1-S1) at dosage of 5×108 CFU, 1×109 CFU and 2×109 CFU respectively. The immunized mice showed no clinic symptom. The results suggested that SL7207 (pVAX1-S1) was safe for mice after oral immunization at dosage of 2×109 CFU or below. BALB/c mice were immunized orally with SL7207 harboring recombinant plasmid at the dosage of 109 and boosted two weeks later with the same dose, for a total of three times. The recombinant Salmonella SL7207(pVAX1-S1) could induce significant humoral immune response in mice compared with the control (P<0.05 or 0.01) at 2 w post-boosting and 2 w post-three immunization. The antibodies against MHV were also detected in small intestinal mucosal samples from immunized mice at 2 w post-three immunization. These results indicated that recombinant SL7207(pVAX1-S1) induced both systemic and local mucosal immunity.
JIAO Hong-mei , JIAO Xin-an , YIN Yue-lan , PAN Zhi-ming , MENG Song-shu
2007, 47(1):131-135.
Abstract:The complete S1 gene from mouse hepatitis virus (MHV) was amplified by RT-PCR and cloned into the pMD18-T vector. After confirmed by the restriction endonuclease analysis and PCR amplification, the positive clone of S1 gene was sequenced and then was transferred into eukaryotic expressing vector pVAX1. The recombinant plasmid pVAX1-S1 was transfected into COS-7 cells. The expressed S1 protein was successfully detected with indirect immunofluorescent assay. Finally, The recombinant plasmid pVAX1-S1 was transformed by electroporation into attenuated Salmonella typhimurium strain SL7207 and confirmed by PCR and Salmonella agglutination test. The recombinant was named as SL7207(pVAX1-S1). 6-week-old BALB/c mice were inoculated orally with SL7207 (pVAX1-S1) at dosage of 5×108 CFU, 1×109 CFU and 2×109 CFU respectively. The immunized mice showed no clinic symptom. The results suggested that SL7207 (pVAX1-S1) was safe for mice after oral immunization at dosage of 2×109 CFU or below. BALB/c mice were immunized orally with SL7207 harboring recombinant plasmid at the dosage of 109 and boosted two weeks later with the same dose, for a total of three times. The recombinant Salmonella SL7207(pVAX1-S1) could induce significant humoral immune response in mice compared with the control (P<0.05 or 0.01) at 2 w post-boosting and 2 w post-three immunization. The antibodies against MHV were also detected in small intestinal mucosal samples from immunized mice at 2 w post-three immunization. These results indicated that recombinant SL7207(pVAX1-S1) induced both systemic and local mucosal immunity.
LI Guang-wei , LIU He , ZHANG Feng , DU Guo-cheng , CHEN Jian
2007, 47(1):136-140.
Abstract:The V2 region of the 16S ribosomal DNA of the ammonia-oxidizing bacteria (AOB) was amplified directly from the environmental sample by using the specific PCR primers. The purified PCR product was cloned into T-vector and was identified as 16S rDNA fragment of AOB by sequencing and Real-time PCR method. Then, the recombined plasmid was used as standard molecule sample in Real-time PCR for AOB quantification. The numbers of the AOB were monitored in samples of both aerobic granular sludge and activated sludge influenced by PCP by using Real-time PCR. The results showed that the numbers of AOB in aerobic granular sludge and activated sludge were 4.28×107±5.44×106cells/g dried sludge and 2.51×109±8.61×108cells/g dried sludge without PCP in the reactors, respectively. With the increase of PCP concentration (from 0mg/L to 50mg/L)，the numbers of AOB in both types of sludge had no obvious change（P>0.05）. The numbers of AOB had no obvious correlation with ammonia removal (P>0.05). The main effect of PCP on AOB in both types of sludge was to inhibit their metabolic activity.
LI Guang-wei , LIU He , ZHANG Feng , DU Guo-cheng , CHEN Jian
2007, 47(1):136-140.
Abstract:The V2 region of the 16S ribosomal DNA of the ammonia-oxidizing bacteria (AOB) was amplified directly from the environmental sample by using the specific PCR primers. The purified PCR product was cloned into T-vector and was identified as 16S rDNA fragment of AOB by sequencing and Real-time PCR method. Then, the recombined plasmid was used as standard molecule sample in Real-time PCR for AOB quantification. The numbers of the AOB were monitored in samples of both aerobic granular sludge and activated sludge influenced by PCP by using Real-time PCR. The results showed that the numbers of AOB in aerobic granular sludge and activated sludge were 4.28×107±5.44×106cells/g dried sludge and 2.51×109±8.61×108cells/g dried sludge without PCP in the reactors, respectively. With the increase of PCP concentration (from 0mg/L to 50mg/L)，the numbers of AOB in both types of sludge had no obvious change（P>0.05）. The numbers of AOB had no obvious correlation with ammonia removal (P>0.05). The main effect of PCP on AOB in both types of sludge was to inhibit their metabolic activity.
YAO Qing-xia , LIU Xin-wen , QIAN Ping , GUO Dong-chun , CHEN Huan-chun
2007, 47(1):141-144.
Abstract:Porcine interferon-gamma (PoIFN-γ) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neor). Using LipofectamineTM, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400μg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-γ gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400μg/mL, 600μg/mL and 800μg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-γ mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV (vesicular stomatitis virus) activity in MDBK (bovine kidney cell) was 1200IU/106cells. In addition, the effect of rPoIFNγ-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-γ has been inserted into retroviral vector and recombinant retrovirus has been successfully packaged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-γ with natural antiviral bioactivity and can inhibit VSV and FMDV.
YAO Qing-xia , LIU Xin-wen , QIAN Ping , GUO Dong-chun , CHEN Huan-chun
2007, 47(1):141-144.
Abstract:Porcine interferon-gamma (PoIFN-γ) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neor). Using LipofectamineTM, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400μg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-γ gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400μg/mL, 600μg/mL and 800μg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-γ mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV (vesicular stomatitis virus) activity in MDBK (bovine kidney cell) was 1200IU/106cells. In addition, the effect of rPoIFNγ-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-γ has been inserted into retroviral vector and recombinant retrovirus has been successfully packaged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-γ with natural antiviral bioactivity and can inhibit VSV and FMDV.
LIN Juan , MA Cheng , LIU Shu-tao , RAO Ping-fan
2007, 47(1):145-149.
Abstract:High performance ion exchange liquid chromatography and laser light scattering instrument were employed to characterize Ralstonia solanacearum in different growth phases. The pure culture of Ralstonia solanacearum was successfully separated into three characteristic fractions. Chromatographic behaviors of Ralstonia solanacearum in lag phase, logarithmic phase and stationary phase were carefully investigated, and their relationships to the cell concentration, pH of fermentation broth and extracellular polysaccharide (EPSⅠ) content of cell surface were analyzed. It was found that the majority of Ralstonia solanacearum cells obtained at 8h culture time could not be absorbed to the resin due to the strong motility of the cells which could apparently overcome the electrostatic interaction. Furthermore, when the mobility of the cells was destroyed by 4% formaldehyde treatment, the prolonged retention time was recorded due to the stronger adsorbility to the resin. On the other hand, the EPSⅠ content of cell surface was determined to increase with the culture time, indicating an increasing interaction between the cells and the resin. EPSⅠ content of three characteristic chromatographic fractions was determined, and it was found that the higher EPSⅠ content led to the longer retention time of the fraction, which confirmed the above-mentioned observation. As a result, it is concluded that the formation of three chromatographic fractions of the pure culture of Ralstonia solanacearum is attributed to bacterial motility and the interaction of EPSⅠ of cell surface with the anionic exchange resin, and the novel method of ion exchange chromatography of the intact bacterial cells can be a useful tool in bacteriological study.
LIN Juan , MA Cheng , LIU Shu-tao , RAO Ping-fan
2007, 47(1):145-149.
Abstract:High performance ion exchange liquid chromatography and laser light scattering instrument were employed to characterize Ralstonia solanacearum in different growth phases. The pure culture of Ralstonia solanacearum was successfully separated into three characteristic fractions. Chromatographic behaviors of Ralstonia solanacearum in lag phase, logarithmic phase and stationary phase were carefully investigated, and their relationships to the cell concentration, pH of fermentation broth and extracellular polysaccharide (EPSⅠ) content of cell surface were analyzed. It was found that the majority of Ralstonia solanacearum cells obtained at 8h culture time could not be absorbed to the resin due to the strong motility of the cells which could apparently overcome the electrostatic interaction. Furthermore, when the mobility of the cells was destroyed by 4% formaldehyde treatment, the prolonged retention time was recorded due to the stronger adsorbility to the resin. On the other hand, the EPSⅠ content of cell surface was determined to increase with the culture time, indicating an increasing interaction between the cells and the resin. EPSⅠ content of three characteristic chromatographic fractions was determined, and it was found that the higher EPSⅠ content led to the longer retention time of the fraction, which confirmed the above-mentioned observation. As a result, it is concluded that the formation of three chromatographic fractions of the pure culture of Ralstonia solanacearum is attributed to bacterial motility and the interaction of EPSⅠ of cell surface with the anionic exchange resin, and the novel method of ion exchange chromatography of the intact bacterial cells can be a useful tool in bacteriological study.
HUA Shao-feng , LI Shu-ben , TAN Hai-dong , ZHAO Jia-zheng
2007, 47(1):150-155.
Abstract:Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71% to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with γ proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSWⅢ of typeⅠmethanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of typeⅠmethanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in typeⅠmethanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.
HUA Shao-feng , LI Shu-ben , TAN Hai-dong , ZHAO Jia-zheng
2007, 47(1):150-155.
Abstract:Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71% to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with γ proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSWⅢ of typeⅠmethanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of typeⅠmethanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in typeⅠmethanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.
MI Shi-jun , BAI Ying-guo , MENG Kun , WANG Ya-ru , YAO Bin
2007, 47(1):156-160.
Abstract:An α-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The α-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The α-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45℃. The enzyme is stable between pH5.0 and 6.0 below 40℃. The α-galactosidase activity is slightly inhibited by Ag+, which is dissimilar to other α-galactosidases. Kinetic studies of the α-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L.min－1.mg－1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the α-galactosidase is a novel one.
MI Shi-jun , BAI Ying-guo , MENG Kun , WANG Ya-ru , YAO Bin
2007, 47(1):156-160.
Abstract:An α-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The α-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The α-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45℃. The enzyme is stable between pH5.0 and 6.0 below 40℃. The α-galactosidase activity is slightly inhibited by Ag+, which is dissimilar to other α-galactosidases. Kinetic studies of the α-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L.min－1.mg－1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the α-galactosidase is a novel one.
SHI Wan-liang , ZHONG Chuan-qi , TANG Bing , SHEN Ping
2007, 47(1):161-163.
Abstract:A halophilic extracellular protease from a halophilic archaea Natrinema sp. R6-5 was purified to SDS-PAGE homogeneity using bacitracin-Sepharose 4B chromatography. A molecular mass of the purified protease subunit was 62KD determined by SDS-PAGE. The protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that the protease belong to serine protease. The protease exhibited optimum NaCl concentration is 3 mol/L. At the 3 mol/L NaCl concentration, the optimum temperature and the optimum pH were 45℃ and 8.0. The protease could keep high activity and stability in high salt environment and had potential application value.
SHI Wan-liang , ZHONG Chuan-qi , TANG Bing , SHEN Ping
2007, 47(1):161-163.
Abstract:A halophilic extracellular protease from a halophilic archaea Natrinema sp. R6-5 was purified to SDS-PAGE homogeneity using bacitracin-Sepharose 4B chromatography. A molecular mass of the purified protease subunit was 62KD determined by SDS-PAGE. The protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that the protease belong to serine protease. The protease exhibited optimum NaCl concentration is 3 mol/L. At the 3 mol/L NaCl concentration, the optimum temperature and the optimum pH were 45℃ and 8.0. The protease could keep high activity and stability in high salt environment and had potential application value.
GAN Yaokun , XU Qian , PENG Guangyin , WU Kai , LIU Kai-yu
2007, 47(1):164-167.
Abstract:To explore the effect of formaldehyde on DNA-protein cross-links (DPC) in eucaryotic cells and prokaryotic cells, Pichia pastoris and E.coli DH5α were chosen as materials to evaluate the amount of DPC induced by liquid formaldehyde in vivo by the method of KCl-SDS assay. The results showed that formaldehyde could not induce DPC at low dose(25μmol/L，P＞0.05), but could obviously induce DPC at higher dose (125 and 625μmol/L，P＜0.05). The DPC coefficient in Pichia pastoris was 10-fold higher than that in E.coli DH5α. It is concluded that formaldehyde could induce DPC in eucaryotic cells and prokaryotic cells with a dose-dependent manner and the DPC coefficient in Pichia pastoris is higher than that in E.coli DH5α.
GAN Yaokun , XU Qian , PENG Guangyin , WU Kai , LIU Kai-yu
2007, 47(1):164-167.
Abstract:To explore the effect of formaldehyde on DNA-protein cross-links (DPC) in eucaryotic cells and prokaryotic cells, Pichia pastoris and E.coli DH5α were chosen as materials to evaluate the amount of DPC induced by liquid formaldehyde in vivo by the method of KCl-SDS assay. The results showed that formaldehyde could not induce DPC at low dose(25μmol/L，P＞0.05), but could obviously induce DPC at higher dose (125 and 625μmol/L，P＜0.05). The DPC coefficient in Pichia pastoris was 10-fold higher than that in E.coli DH5α. It is concluded that formaldehyde could induce DPC in eucaryotic cells and prokaryotic cells with a dose-dependent manner and the DPC coefficient in Pichia pastoris is higher than that in E.coli DH5α.
SONG Zhong-bang , CHEN Li-mei , LI Kun-zhi , PAN Zheng-bo
2007, 47(1):168-172.
Abstract:Ribulose monophosphate pathway (RuMP), which was originally found in methylotrophic bacteria, is now recognized as a metabolic pathway widespread in most bacteria and involved in formaldehyde assimilation and detoxification. 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of this pathway. This review describes the physiological significance of RuMP pathway derived from a variety of bacteria, the organizations and expressional regulations of HPS and PHI genes and the perspectives for applications of the two genes.
SONG Zhong-bang , CHEN Li-mei , LI Kun-zhi , PAN Zheng-bo
2007, 47(1):168-172.
Abstract:Ribulose monophosphate pathway (RuMP), which was originally found in methylotrophic bacteria, is now recognized as a metabolic pathway widespread in most bacteria and involved in formaldehyde assimilation and detoxification. 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of this pathway. This review describes the physiological significance of RuMP pathway derived from a variety of bacteria, the organizations and expressional regulations of HPS and PHI genes and the perspectives for applications of the two genes.
HONG Yi-guo , GUO Jun , SUN Guo-ping
2007, 47(1):173-177.
Abstract:The discovery of Electricigens substantially changed the meaning of Microbial Fuel Cell (MFC) and exhibited a broad prospect for application. This kind microorganism can completely oxidize organic compounds with electrode as sole electron acceptor and then transfer the electrons derived from that oxidation onto the anode of a MFC through electron transport chain. When the electrons transfer from anode to cathode, the current was generated continuously. At same time, they gain energy to support their growth from the electron transport. The biochemical metabolism process was considered as a new type microbiological respiration. Based on the new concept, MFC offered the possibility of efficient treatment waste-water and generation electricity simultaneously, which would fuel the waste-water into a profitable industry in the future. So the application of MFC in the waste-water treatment would be most promising.
HONG Yi-guo , GUO Jun , SUN Guo-ping
2007, 47(1):173-177.
Abstract:The discovery of Electricigens substantially changed the meaning of Microbial Fuel Cell (MFC) and exhibited a broad prospect for application. This kind microorganism can completely oxidize organic compounds with electrode as sole electron acceptor and then transfer the electrons derived from that oxidation onto the anode of a MFC through electron transport chain. When the electrons transfer from anode to cathode, the current was generated continuously. At same time, they gain energy to support their growth from the electron transport. The biochemical metabolism process was considered as a new type microbiological respiration. Based on the new concept, MFC offered the possibility of efficient treatment waste-water and generation electricity simultaneously, which would fuel the waste-water into a profitable industry in the future. So the application of MFC in the waste-water treatment would be most promising.
2007, 47(1):178-181.
Abstract:The general projects on subdisciplines of Microbiology which were funded by the National Natural Science Foundation of China (NSFC) during the financial years of 2000 to 2006 are reviewed in this article. The number of general projects and funds involved are analyzed to give an overview of fundamental research in microbiology in China. Some important research fields in microbiology that the NSFC should support in the future are also recommended.
2007, 47(1):178-181.
Abstract:The general projects on subdisciplines of Microbiology which were funded by the National Natural Science Foundation of China (NSFC) during the financial years of 2000 to 2006 are reviewed in this article. The number of general projects and funds involved are analyzed to give an overview of fundamental research in microbiology in China. Some important research fields in microbiology that the NSFC should support in the future are also recommended.
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