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ABSTRACT: BACKGROUND: Aminopeptidases have great application in the food industry. Current researches on the expression of aminopeptidases mainly focus on the Escherichia coli expression system. However, the application of recombinant E. coli in the food industry is restricted due to its pathogenicity and low secretory efficiency, which should be concerned in the industrial production of aminopeptidases. RESULTS: The gene of aminopeptidase from Bacillus subtilis Zj016 (BSAP) was identified. Overexpression and secretion of BSAP were achieved in a B. subtilis expression system with the signal peptide of itself. The yield researched 52±1.9 U mL(-1) , which was 18 times that of wild-type microbe. The purified enzyme was stable at pH 7.5-9.0 and below 60°C, and was inhibited by several metal ions except appropriate Co(2+) . BSAP was most active toward p-nitroaniline derivatives of Leu, Arg and Lys. Homology modeling and structure analysis showed that there was a flexible protease-associated domain in the predicted structure of BSAP. CONCLUSIONS: The study presented a simple procedure for overexpression and purification of BSAP. The substrate specificity and structure information were indicated based on the characterization and homology modeling. This will be useful for further research of aminopeptidases not only from an academic standpoint but also from an applied point of view.
Journal of the Science of Food and Agriculture 02/2013; · 1.44 Impact Factor
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Wei Liu,
Shaobo Xiao,
Mao Li,
Shaohua Guo,
Sha Li,
Rui Luo,
Zhixin Feng,
Bin Li, Zhemin Zhou,
Guoqing Shao,
Huanchun Chen,
Liurong Fang
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ABSTRACT: BACKGROUND: Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses. RESULTS: We performed the first comprehensive analysis of M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins (P97, P102, P146, P159, P216, and LppT), cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence. CONCLUSIONS: We studied the virulence attenuation mechanism of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form the foundation of future investigations into the pathogenesis of M. hyopneumoniae and facilitate the design of new vaccines.
BMC Genomics 02/2013; 14(1):80. · 4.07 Impact Factor
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ABSTRACT: The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k (cat)/K (m) was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.
Biotechnology Letters 01/2013; · 1.68 Impact Factor
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ABSTRACT: BACKGROUND: Transglutaminase (TGase) is secreted as a zymogen (Pro-TGase) and is then processed by removal of its N-terminal region through exogenous proteolytic activity. In this study it was discovered that the Pro-TGase from Streptomyces hygroscopicus was also activated by its TGase (processed through exogenous proteolytic activity), resulting in a different active form. RESULTS: The two TGases exhibited different ionic strengths, hydrophobicities, K(m) values and stabilities. Circular dichroism spectral analysis showed that the two enzymes had non-identical secondary structures, while liquid chromatography/mass spectrometry (LC-MS) analysis indicated that they differed in molecular mass by 111 Da. The formation of the TGase activated from Pro-TGase by TGase was delayed compared with that of TGase processed through exogenous proteolytic activity. Furthermore, it was found that the TGase activated from Pro-TGase by TGase did not activate Pro-TGase. CONCLUSION: Two TGases derived from the same zymogen from S. hygroscopicus were discovered. These two active forms of TGase may be due to different activation processes: one of them is catalysed by its own active TGase, while the other is activated by an exogenous protease. © 2013 Society of Chemical Industry.
Journal of the Science of Food and Agriculture 10/2012; · 1.44 Impact Factor
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ABSTRACT: Flagella contribute to the virulence of bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellar phase variation is believed to facilitate bacterial evasion of the host immune response. In this study, the flnA gene that encodes Escherichia coli H17 flagellin was examined by whole genome sequencing and genetic deletion analysis. Unilateral flagellar phase variation has been reported in E. coli H3, H47 and H17 strains, although the mechanism for phase variation in the H17 strain has not been previously understood. Analysis of phase variants indicated that the flagellar phase variation in the H17 strain was caused by the deletion of an ∼35 kb DNA region containing the flnA gene from diverse excision sites. The presence of covalently closed extrachromosomal circular forms of this excised 35 kb region was confirmed by the two-step polymerase chain reaction. The deletion and complementation test revealed that the Int1157 integrase, a tyrosine recombinase, mediates the excision of this region. Unlike most tyrosine recombinases, Int1157 is suggested to recognize diverse sites and mediate recombination between non-homologous DNA sequences. This is the first report of non-homologous recombination mediating flagellar phase variation.
Nucleic Acids Research 01/2012; 40(10):4530-8. · 8.03 Impact Factor
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ABSTRACT: Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of <β-subunit> <α-subunit> , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of <α-subunit> <β-subunit> , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)(2)] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)(2) and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)(2) but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a <β-subunit> <α-subunit> order. Our findings expand the general features of self-subunit swapping maturation.
PLoS ONE 01/2012; 7(11):e50829. · 4.09 Impact Factor
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Wei Liu,
Liurong Fang,
Mao Li,
Sha Li,
Shaohua Guo,
Rui Luo,
Zhixin Feng,
Bin Li, Zhemin Zhou,
Guoqing Shao,
Huanchun Chen,
Shaobo Xiao
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ABSTRACT: Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages.
PLoS ONE 01/2012; 7(4):e35698. · 4.09 Impact Factor
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Yanwen Xiong,
Ping Wang,
Ruiting Lan,
Changyun Ye,
Hua Wang,
Jun Ren,
Huaiqi Jing,
Yiting Wang, Zhemin Zhou,
Xuemei Bai,
Zhigang Cui,
Xia Luo,
Ailan Zhao,
Yan Wang,
Shaomin Zhang,
Hui Sun,
Lei Wang,
Jianguo Xu
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ABSTRACT: An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.
PLoS ONE 01/2012; 7(4):e36144. · 4.09 Impact Factor
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ABSTRACT: Streptomyces transglutaminase (TGase) is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli.
A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+). Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties.
Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.
Microbial Cell Factories 12/2011; 10:112. · 3.55 Impact Factor
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ABSTRACT: Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E. coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E. coli.
FEMS Microbiology Letters 11/2011; 324(2):98-105. · 2.04 Impact Factor
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ABSTRACT: Bacillus megaterium, an industrial strain, has been widely used in protein production and the vitamin C industry. Here we reported a finished, annotated, and compared 4.14-Mbp high-quality genome sequence of B. megaterium WSH-002, which is the companion strain for Ketogulonicigenium vulgare in the vitamin C industry and is stocked in our laboratory.
Journal of bacteriology 11/2011; 193(22):6389-90. · 3.94 Impact Factor
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ABSTRACT: Ketogulonicigenium vulgare is an industrial organism commonly used in the vitamin C industry. Here, we report the finished, annotated, and compared 3.28-Mbp high-quality genome sequence of Ketogulonicigenium vulgare WSH-001, a 2-keto-l-gulonic acid-producing industrial strain stocked in our laboratory.
Journal of bacteriology 11/2011; 193(21):6108-9. · 3.94 Impact Factor
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ABSTRACT: Aeromonas veronii strain B565 was isolated from aquaculture pond sediment in China. We present here the complete genome sequence of B565 and compare it with 2 published genome sequences of pathogenic strains in the Aeromonas genus. The result represents an independent stepwise acquisition of virulence factors of pathogenic strains in this genus.
Journal of bacteriology 07/2011; 193(13):3389-90. · 3.94 Impact Factor
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Zhihong Sun,
Xia Chen,
Jicheng Wang,
Wenjing Zhao,
Yuyu Shao,
Zhuang Guo,
Xingchang Zhang, Zhemin Zhou,
Tiansong Sun,
Lei Wang,
He Meng,
Heping Zhang,
Wei Chen
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ABSTRACT: Lactobacillus delbrueckii subsp. bulgaricus strain ND02 is a Chinese commercial dairy starter used for the manufacture of yoghurt. It was isolated from naturally fermented yak milk in Qinghai, China. Here, we report the main genome features of ND02 and several differences with two other published genomes of Lactobacillus delbrueckii subsp. bulgaricus strains.
Journal of bacteriology 07/2011; 193(13):3426-7. · 3.94 Impact Factor
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ABSTRACT: Bordetella pertussis is the causative agent of pertussis. Here, we report the genome sequence of Bordetella pertussis strain CS, isolated from an infant patient in Beijing and widely used as a vaccine strain for production of an acellular pertussis vaccine in China.
Journal of bacteriology 05/2011; 193(15):4017-8. · 3.94 Impact Factor
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ABSTRACT: Lactobacillus helveticus strain H10 was isolated from traditional fermented milk in Tibet, China. We sequenced the whole genome of strain H10 and compared it to the published genome sequence of Lactobacillus helveticus DPC4571.
Journal of bacteriology 03/2011; 193(10):2666-7. · 3.94 Impact Factor
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ABSTRACT: Yersinia enterocolitica is a heterogeneous bacterial species with a wide range of animal reservoirs through which human intestinal illness can be facilitated. In contrast to the epidemiological pattern observed in the United States, infections in China present a pattern similar to those in European countries and Japan, wherein "Old World" strains (biotypes 2 to 5) are prevalent. To gain insights into the evolution of Y. enterocolitica and pathogenic properties toward human hosts, we sequenced the genome of a biotype 3 strain, 105.5R(r) (O:9), obtained from a Chinese patient. Comparative genome sequence analysis with strain 8081 (1B/O:8) revealed new insights into Y. enterocolitica. Both strains have more than 14% specific genes. In strain 105.5R(r), putative virulence factors were found in strain-specific genomic pathogenicity islands that comprised a novel type III secretion system and rtx-like genes. Many of the loci representing ancestral clusters, which are believed to contribute to enteric survival and pathogenesis, are present in strain 105.5R(r) but lost in strain 8081. Insertion elements in 105.5R(r) have a pattern distinct from those in strain 8081 and were exclusively located in a strain-specific region. In summary, our comparative genome analysis indicates that these two strains may have attained their pathogenicity by completely separate evolutionary events, and the 105.5R(r) strain, a representative of the Old World biogroup, lies in a branch of Y. enterocolitica that is distinct from the "New World" 8081 strain.
Journal of clinical microbiology 02/2011; 49(4):1251-9. · 4.16 Impact Factor
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Wei Liu,
Zhixin Feng,
Liurong Fang, Zhemin Zhou,
Qiang Li,
Sha Li,
Rui Luo,
Lei Wang,
Huanchun Chen,
Guoqing Shao,
Shaobo Xiao
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ABSTRACT: Mycoplasma hyopneumoniae strain 168, a pathogenic strain prevalent in China, was isolated in 1974. Although this strain has been widespread for a long time, the genome sequence had not been determined. Here, we announce the complete genome sequence of M. hyopneumoniae strain 168.
Journal of bacteriology 02/2011; 193(4):1016-7. · 3.94 Impact Factor
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Zhihong Sun,
Xia Chen,
Jicheng Wang,
Wenjing Zhao,
Yuyu Shao,
Lan Wu, Zhemin Zhou,
Tiansong Sun,
Lei Wang,
He Meng,
Heping Zhang,
Wei Chen
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ABSTRACT: Streptococcus thermophilus strain ND03 is a Chinese commercial dairy starter used for the manufacture of yogurt. It was isolated from naturally fermented yak milk in Qinghai, China. We present here the complete genome sequence of ND03 and compare it to three other published genomes of Streptococcus thermophilus strains.
Journal of bacteriology 02/2011; 193(3):793-4. · 3.94 Impact Factor
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Ping Wang,
Yanwen Xiong,
Ruiting Lan,
Changyun Ye,
Hua Wang,
Jun Ren,
Huaiqi Jing,
Yiting Wang, Zhemin Zhou,
Zhigang Cui, [......],
Dong Jin,
Xuemei Bai,
Ailan Zhao,
Yan Wang,
Shaomin Zhang,
Hui Sun,
Juan Li,
Tao Wang,
Lei Wang,
Jianguo Xu
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ABSTRACT: In addition to the large virulence plasmid pO157, a novel 38-kb conjugative plasmid, pO157_Sal, was identified and sequenced from an Escherichia coli O157:H7 outbreak-associated Chinese isolate that shares high similarity with a plasmid in Salmonella enterica serovar Agona. The plasmid was found in 15 of 326 isolates, 12 of which were of the same pulsed-field gel electrophoresis type.
Journal of clinical microbiology 02/2011; 49(4):1594-7. · 4.16 Impact Factor
