溶藻弧菌Ⅲ型分泌系统vscP基因的克隆与生物信息学分析
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摘要
根据NCBI上登录的溶藻弧菌(Vibrio alginolyticus)全基因组序列,设计一对克隆溶藻弧菌HY9901株Ⅲ型分泌系统"分子尺"蛋白VscP全长基因的特异性引物,PCR扩增获得基因的全长片段。结果显示,vsc P(Gen Bank登录号FR780678)开放阅读框ORF为1 206 bp,共编码401个氨基酸残基。根据推导的氨基酸序列预测其分子质量约为44.4 ku,等电点为5.72。Signal 4.1 Server和TMHMM Server 2.0预测结果表明,VscP无信号肽与跨膜结构域。Soft Berry-Psite预测结果显示,VscP含有3个N端糖基化位点,2个cAMP及c GMP依赖性蛋白激酶磷酸化位点,5个蛋白激酶C磷酸化位点,9个酪蛋白激酶II磷酸化位点,2个N端豆蔻酰基化位点,1个酰胺化位点和3个微体C末端靶信号位点。运用MEGA6.0构建的VscP系统进化树显示,溶藻弧菌VscP与副溶血弧菌(V.parahaemolyticus)聚为一簇。应用SWISS-MODEL软件构建VscP亚基三维结构模型发现,其与鞭毛Fil K蛋白有相似构型。
Primers for PCR cloning were designed according to the whole genome sequence of Vibrio alginolyticus published in Gen Bank. The type 3 secretion system(T3SS) Yop secretion protein P(VscP) gene of V. alginolyticus strain HY9901 was amplified by PCR and cloned into p MD 18-T vector. Sequence analysis reveals that the ORF of VscP gene(Gen Bank Accession: FR780678) is 1 206 bp and eneodes a putative protein of 401 amino acids. The predicted molecular weight(MW) of VscP was 44.4 ku with an estimated p I of 5.72. Using Signal Server 4.1 and TMHMM Server 2.0 software, and it was predieted that the VscP protein did not contain a signal peptide or a transmembranous region. The VscP protein had three N-glycosylation sites, two c AMP and c GMP dependent protein kinase phosphorylation site, five protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, two N-myristoylation sites, one amidation site and three loci microbody C-terminal targeting signal predieted by Soft Berry-Psite software. This protein shows high genetic relationship with Vibrio parahaemolyticus via a molecular phylogenetic tree constructed by MEGA6.0 software. A model of VscP subunits application of SWISS-MODEL work-space is built and its three-dimensional structure is found to have similar configuration with flagellar hook-length control protein Fli K.
Primers for PCR cloning were designed according to the whole genome sequence of Vibrio alginolyticus published in Gen Bank. The type 3 secretion system(T3SS) Yop secretion protein P(VscP) gene of V. alginolyticus strain HY9901 was amplified by PCR and cloned into p MD 18-T vector. Sequence analysis reveals that the ORF of VscP gene(Gen Bank Accession: FR780678) is 1 206 bp and eneodes a putative protein of 401 amino acids. The predicted molecular weight(MW) of VscP was 44.4 ku with an estimated p I of 5.72. Using Signal Server 4.1 and TMHMM Server 2.0 software, and it was predieted that the VscP protein did not contain a signal peptide or a transmembranous region. The VscP protein had three N-glycosylation sites, two c AMP and c GMP dependent protein kinase phosphorylation site, five protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, two N-myristoylation sites, one amidation site and three loci microbody C-terminal targeting signal predieted by Soft Berry-Psite software. This protein shows high genetic relationship with Vibrio parahaemolyticus via a molecular phylogenetic tree constructed by MEGA6.0 software. A model of VscP subunits application of SWISS-MODEL work-space is built and its three-dimensional structure is found to have similar configuration with flagellar hook-length control protein Fli K.
引文
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[15]WOOD S E,JIN J,LLOYD S A.Ysc P and Ysc U switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein Ysc I[J].Journal of Bacteriology,2008,190(12):4252-4262.
[16]GALLE M,CARPENTIER I,BEYAERT R.Structure and function of the Type III secretion system of Pseudomonas aeruginosa[J].Current Protein and Peptide Science,2012,13(8):831-842.
[17]RüSSMANN H,KUBORI T,SAUER J,et al.Molecular and functional analysis of the type III secretion signal of the Salmonella enterica Inv J protein[J].Molecular Microbiology,2002,46(3):769-779.
[18]MAGDALENA J,HACHANI A,CHAMEKH M,et al.Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins[J].Journal of Bacteriology,2002,184(13):3433-3441.
[19]BERGERON J R,FERNANDEZ L,WASNEY G A,et al.The structure of a type 3 secretion system(T3SS)ruler protein suggests a molecular mechanism for needle length sensing[J].The Journal of Biological Chemistry,2016,291(4):1676-1691.
[20]AGRAIN C,SORG I,PAROZ C,et al.Secretion of Ysc P from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity[J].Molecular Microbiology,2005,57(5):1415-1427.
[21]BLOCKER A,KOMORIYA K,AIZAWA S-I.Type III secretion systems and bacterial flagella:insights into their function from structural similarities[J].Proceedings of the National Academy of Sciences,2003,100(6):3027-3030.
[22]GALKIN V E,SCHMIED W H,SCHRAIDT O,et al.The structure of the Salmonella typhimurium type III secretion system needle shows divergence from the flagellar system[J].Journal of Molecular Biology,2010,396(5):1392-1397.
[23]RIORDAN K E,SORG J A,BERUBE B J,et al.Impassable Ysc P substrates and their impact on the Yersinia enterocolitica type III secretion pathway[J].Journal of Bacteriology,2008,190(18):6204-6216.
[24]MUKERJEA R,GHOSH P.Functionally essential interaction between Yersinia Ysc O and the T3S4 domain of Ysc P[J].Journal of Bacteriology,2013,195(20):4631-4638.
[2]赖迎迢,陶家发,孙承文,等.鱼源溶藻弧菌生物学特性和病理组织学观察[J].微生物学报,2014,54(11):1378-1384.
[3]SANTHYIA A V,MULLOORPEEDIKAYIL R G,KOLLANOOR R J,et al.Molecular variations in Vibrio alginolyticus and V.harveyi in shrimp-farming systems upon stress[J].Brazilian Journal of Microbiology,2015,46(4):1001-1008.
[4]SPERLING L,ALTER T,HUEHN S.Prevalence and Antimicrobial Resistance of Vibrio spp.in Retail and Farm Shrimps in Ecuador[J].J Food Prot,2015,78(11):2089-2092.
[5]苑淑宾,朱爱意.溶藻弧菌对水产动物致病性及其防治的研究进展[J].浙江海洋学院学报(自然科学版),2012,31(3):256-264.
[6]ZHAO Z,CHEN C,HU C Q,et al.The type III secretion system of Vibrio alginolyticus induces rapid apoptosis,cell rounding and osmotic lysis of fish cells[J].Microbiology,2010,156(9):2864-2872.
[7]ZHAO Z,Zhang L P,REN C H,et al.Autophagy is induced by the type III secretion system of Vibrio alginolyticus in several mammalian cell lines[J].Archives of Microbiology,2011,193(1):53-61.
[8]程海燕,庞欢瑛,鲁义善,等.溶藻弧菌acf A基因克隆与生物信息学分析[J].广东海洋大学学报,2014,34(1):9-14.
[9]ZHOU Z J,PANG H Y,DING Y,et al.Vsc O,a putative T3SS chaperone escort of Vibrio alginolyticus,contributes to virulence in fish and is a target for vaccine development[J].Fish&Shellfish Immunology,2013,35(5):1523-1531.
[10]王青柏,胡超群,陈偿,等.溶藻弧菌Ⅲ型分泌系统vsc C基因缺失株的构建[J].安徽农业科学,2012,13(3):492-496.
[11]AGRAIN C,CALLEBAUT I,JOURNET L,et al.Characterization of a Type III secretion substrate specificity switch(T3S4)domain in Ysc P from Yersinia enterocolitica[J].Molecular Microbiology,2005,56(1):54-67.
[12]BüTTNER D,BONAS U.Port of entry–the type III secretion translocon[J].Trends in Microbiology,2002,10(4):186-192.
[13]COOMBES B K,FINLAY B B.Insertion of the bacterial type III translocon:not your average needle stick[J].Trends Microbiol,2005,13(3):92-95.
[14]MONJARAS FERIA J,GARCIA-GOMEZ E,ESPINOSA N,et al.Role of Esc P(Orf16)in injectisome biogenesis and regulation of type III protein secretion in enteropathogenic Escherichia coli[J].Journal of Bacteriology,2012,194(22):6029-6045.
[15]WOOD S E,JIN J,LLOYD S A.Ysc P and Ysc U switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein Ysc I[J].Journal of Bacteriology,2008,190(12):4252-4262.
[16]GALLE M,CARPENTIER I,BEYAERT R.Structure and function of the Type III secretion system of Pseudomonas aeruginosa[J].Current Protein and Peptide Science,2012,13(8):831-842.
[17]RüSSMANN H,KUBORI T,SAUER J,et al.Molecular and functional analysis of the type III secretion signal of the Salmonella enterica Inv J protein[J].Molecular Microbiology,2002,46(3):769-779.
[18]MAGDALENA J,HACHANI A,CHAMEKH M,et al.Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins[J].Journal of Bacteriology,2002,184(13):3433-3441.
[19]BERGERON J R,FERNANDEZ L,WASNEY G A,et al.The structure of a type 3 secretion system(T3SS)ruler protein suggests a molecular mechanism for needle length sensing[J].The Journal of Biological Chemistry,2016,291(4):1676-1691.
[20]AGRAIN C,SORG I,PAROZ C,et al.Secretion of Ysc P from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity[J].Molecular Microbiology,2005,57(5):1415-1427.
[21]BLOCKER A,KOMORIYA K,AIZAWA S-I.Type III secretion systems and bacterial flagella:insights into their function from structural similarities[J].Proceedings of the National Academy of Sciences,2003,100(6):3027-3030.
[22]GALKIN V E,SCHMIED W H,SCHRAIDT O,et al.The structure of the Salmonella typhimurium type III secretion system needle shows divergence from the flagellar system[J].Journal of Molecular Biology,2010,396(5):1392-1397.
[23]RIORDAN K E,SORG J A,BERUBE B J,et al.Impassable Ysc P substrates and their impact on the Yersinia enterocolitica type III secretion pathway[J].Journal of Bacteriology,2008,190(18):6204-6216.
[24]MUKERJEA R,GHOSH P.Functionally essential interaction between Yersinia Ysc O and the T3S4 domain of Ysc P[J].Journal of Bacteriology,2013,195(20):4631-4638.