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1 微生物学通报 Microbiology China Nov. 20, 2016, 43(11): DOI: /j.microbiol.china 研究报告推定第四类羊毛硫素合成酶生物信息学分析胡彦婷 1, 安丽康尹淑丽程辉彩张根伟张丽萍刘洪伟 ( ) ( ) ( ) * 摘要 : 目的获得更多关于第四类羊毛硫素合成酶的序列及蛋白结构特征信息, 并为研究其作用机制及生物学功能奠定基础方法应用多种软件分析和预测了山丘链霉菌枯草芽孢杆菌肺炎双球菌惰性乳杆菌德氏乳杆菌长双歧杆菌拟无枝酸菌大芬戈尔德菌中此类蛋白质的理化性质结构域二级结构等, 同时采用邻位连接法对这 8 种蛋白及其结构域进行了进化树的构建结果所研究的这 8 种蛋白均为亲水性蛋白, 均无信号肽 ; 山丘链霉菌枯草芽孢杆菌肺炎双球菌德氏乳杆菌长双歧杆菌拟无枝酸菌大芬戈尔德菌的此类合成酶属于酸性蛋白, 惰性乳杆菌的类第四类羊毛硫素合成酶属于碱性蛋白 ; 除山丘链霉菌枯草芽孢杆菌肺炎双球菌的此类合成酶不稳定外, 其它菌株的合成酶均稳定 ; 进化结果表明枯草芽孢杆菌和肺炎链球菌同源性最高,LANC-like 结构域与合成酶的进化关系保持了高度的一致, 而 STYKc/S_TKc 结构域的进化关系表现出了一定的差异 ; 二级结构主要以 α 螺旋和无规卷曲为主 ; 所有的蛋白均有 LANC-like 结构域结论类第四类羊毛硫素合成酶在不同的菌种中具有一定的保守性, 因此能够发挥相似的生物学功能研究结果对进一步研究第四类羊毛硫素合成酶具有一定的参考价值, 尤其是为通过该途径进一步提高枯草芽孢杆菌生防价值提供基础关键词 : 类第四类羊毛硫素合成酶, 生物信息学, 枯草芽孢杆菌 Bioinformatics to predict type IV lanthipeptide synthetases HU Yan-Ting 1, AN Li-Kang YIN Shu-Li CHENG Hui-Cai ZHANG Gen-Wei ZHANG Li-Ping LIU Hong-Wei * (1. Hebei University of Technology, Tianjin , China) (2. Institute of Biology, Hebei Academy of Science, Shijiazhuang, Hebei , China) (3. Main Crops Disease of Microbial Control Engineering Technology Research Center in Hebei Province, Shijiazhuang, Hebei , China) Foundation item: Key Basic Research Project in Hebei Province (No D); Key Projects of Hebei Academy of Sciences (No ); Science and Technology Cooperation and Development Funding Projects of Hebei Province (No. Y-15) *Corresponding author: Fax: ; lhwei1987@126.com Received: December 22, 2015; Accepted: March 11, 2016; Published online ( April 05, 2016 基金项目 : (No D) (No ) (No. Y-15) * 通讯作者 :Fax lhwei1987@126.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : 2465 Abstract: [Objective] This study aimed to get more information about sequence and protein structure of type IV lanthipeptide synthetases. [Methods] Eight potential type IV lanthipeptide synthetases were chosen from Bacillus subtilis, Streptomyces collinus Tu 365, Lactobacillus iners LactinV, Streptococcus pneumoniae, Lactobacillus delbruecki, Bifidobacterium longum, Amycolatopsis azurea and Finegoldia magna, and their physicochemical properties, domains, secondary structures were analyzed and predicted using different softwares. By using the Neighbor-joining method of Molecular Evolutionary Genetics Analysis software, a dendrogram was obtained based on genetic distances. [Results] All eight proteins were hydrophilicity and there was no signal peptide in them. The proteins in S. collinus Tu 365, B. subtilis, S. pneumoniae, Lactobacillus delbruecki, Bifidobacterium longum, Amycolatopsis azurea and Finegoldia magna were acidic whereas the others were alkaline. The proteins in S. collinus Tu 365, B. subtilis and S. pneumoniae were unstable whereas the others were stable. The evolutionary analysis showed that B. subtilis had the closest genetic relationship with S. pneumonia. The evolutionary relationships of LANC-like domains were similar to total synthase, which was different from STYKc/S_TKc domains. Alpha helix and coil were the basic secondary structure. All those proteins contained LANC-like domain. [Conclusion] All that eight synthetases have their conservative structure in different bacteria, therefore, they can play the similar biological functions. All these results will provide information of the type IV lanthipeptide synthetase for further study, especially for improving the bio-control value of B. subtilis. Keywords: The predicted type IV lanthipeptide synthetase, Bioinformatics, Bacillus subtilis [1] [2] [3] LanB LanC LanM 3 N C [3-5] (LanL enzymes) N C LanC-like [3-5] [6-8] [9] XF-1 (Bacillus subtilis XF-1) (Type IV lanthipeptide synthetase like gene T4LSL)

3 2466 微生物学通报 Microbiol. China 2016, Vol.43, No.11 1 材料与方法 1.1 类第四类羊毛硫素合成酶序列的获取与确定 NCBI ( XF-1 (Bacillus subtilis XF-1) (CP ) [10] Bagel [11] ( 1 NCBI Iftime [2] (Streptomyces collinus Tu 365) Type IV lanthipeptide synthetase (AGS73699) 1.2 类第四类羊毛硫素合成酶的理化性质分析 Prot Param [12] ( org/protparam/) SignalP [13] ( Organism group Gram-negative bacteria 1.3 激酶磷酸化修饰位点预测 KinasePhos [14] ( mbc.nctu.edu.tw/) Serine (S) Kinase Threonine (T) Kinase Tyrosine (Y) Kinase Select All Phosphohistidine (H) 1.4 二级结构及结构域预测 PSIPRED [15] ( cs.ucl.ac.uk/psipred/) SOPMA [16] ( ibcp.fr/cgi-bin/npsa_automat.pl?page=/npsa/npsa_so pma.html/)psipred SOPMA Smart [17] ( ode.cgi?genomic=1/) DomainDraw [18] ( imb.uq.edu.au/)general Settings 1.5 进化树的建立 BioEdit ClustalW Options MEGA 5.22 [19] (Neighbor-Joining NJ) (Test of Phylogeny) Bootstrap method (Replication) 蛋白质三级结构预测 SWISS-MODEL [20] ( expasy.org/) PDF VMD [21] 2 结果与分析 2.1 类第四类羊毛硫素合成酶序列的获取与确定 NCBI XF-1 Bagel 1 NCBI 500

4 : 2467 图 1 Figure 1 枯草芽孢杆菌 XF-1 中推定的第四类羊毛硫素 Putative lanthipeptide class IV in Bacillus subtilis XF-1 (Lactobacillus iners LactinV) (Streptococcus pneumoniae) (Lactobacillus delbrueckii) (Bifidobacterium longum) (Amycolatopsis azurea) (Finegoldia magna) 部分菌株中类羊毛硫素合成酶的理化性质 ExPASy ProtParam 2 8 Table 1 Species Streptomyces collinus Tu 365 Bacillus subtilis XF-1 Lactobacillus iners LactinV Streptococcus pneumoniae Lactobacillus delbrueckii Bifidobacterium longum Amycolatopsis azurea Finegoldia magna 表 1 部分细菌中类第四类羊毛硫素合成酶一致性比较 Homology comparison of the predicted type IV lanthipeptide synthetases from some bacteria Protein Type IV lanthipeptide synthetase E Protein GenBank Identity Query E value abbreviations accession No. (%) coverage (%) SCLan AGS e-52 Threonine protein kinase BSLan AGE Lanthionine synthetase C-like protein LILan EFO e-127 Lantibiotic synthetase SPLan COO Lanthionine synthetase C-like family protein Lanthionine synthetase C family protein Lanthionine biosynthesis protein LanL Lanthionine synthetase C-like protein Lan.. Note: Proteins are named by initials of genus name and specific name plus Lan. The same as below. LDLan EPB e-114 BLLan ACJ e-91 AALan WP_ e -100 FMLan WP_ e-154

5 2468 微生物学通报 Microbiol. China 2016, Vol.43, No.11 表 2 部分细菌中类第四类羊毛硫肽合成酶理化性质 Table 2 The physical and chemical properties of the predicted type IV lanthipeptide synthetases from some bacteria Signal Number of Molecular Instability Species Protein Theoretical pi Hydropathicity peptide amino acids weight (Da) index SCLan NO Streptomyces collinus Tu 365 BSLan NO Bacillus subtilis XF-1 LILan NO Lactobacillus iners LactinV SPLan NO Streptococcus pneumoniae LDLan NO Lactobacillus delbrueckii BLLan NO Bifidobacterium longum AALan NO Amycolatopsis azurea Finegoldia magna FMLan NO SCLan LDLan 类第四类羊毛硫素合成酶的分子进化分析 BioEdit 8 MEGA LANC-like STYKc/S_TKc LANC-like LANC-like STYKc/S_TKc LANC-like

6 : 2469 图 2 8 种菌株中 ( 类 ) 羊毛硫素合成酶的进化关系 Figure 2 Phylogenetic analysis of eight predicted lanthipeptide synthetases from some bacteria Bootstrap (>70 Bootstrap ) (0.1 10%). Note: The numbers in parenthesis represent GenBank accession numbers; The numbers on each branch indicate the bootstrap values (believable if >70); Scale represents the nucleotide changes in per unit (0.1 means 10% changes were observed between two sequences). 图 3 8 种菌株中 STYKc/S_TKc 结构域的进化关系 Figure 3 Phylogenetic analysis of STYKc/S_TKc domain from some bacteria Bootstrap (>70 Bootstrap ) (0.1 10%). Note: The numbers in parenthesis represent GenBank accession numbers; The numbers on each branch indicate the bootstrap values (believable if >70); Scale represents the nucleotide changes in per unit (0.1 means 10% changes were observed between two sequences). 图 4 8 种菌株中 LANC-like 结构域的进化关系 Figure 4 Phylogenetic analysis of LANC-like domain from some bacteria Bootstrap (>70 Bootstrap ) (0.1 10%). Note: The numbers in parenthesis represent GenBank accession numbers; The numbers on each branch indicate the bootstrap values (believable if >70); Scale represents the nucleotide changes in per unit (0.1 means 10% changes were observed between two sequences). 2.4 激酶磷酸化修饰位点预测 KinasePhos 3 8 LDLan 8 3 SCLan LILan LDLan AALan 1:1 4 2:1 BLLan SCLan FMLan LanM

7 2470 微生物学通报 Microbiol. China 2016, Vol.43, No.11 表 3 激酶磷酸化修饰位点汇总 Table 3 The summary of kinase phosphorylation sites Protein Serine Threonine Tyrosine Histidine SCLan BSLan LILan SPLan LDLan BLLan AALan FMLan (pser) (pthr) β Dha/Dhb Lan/MeLan MeLan Lan [2-5,22] / / pser/pthr ATP [23] / 2.5 二级结构及结构域预测 α β β PSIPRED SOPMA 4 α 50.42% α β β 13.87% 28% 8 Smart DomainDraw LANC-like BLLan LANC_like 7 LANC_like LANC-like C- 表 4 部分菌株中类羊毛硫素合成酶二级结构所占百分比 Table 4 The percentage of predicted secondary structure of predicted lanthipeptide synthetases from some bacteria (%) α β β Protein α-helix Coil β-turn β-strand SCLan BSLan LILan SPLan LDLan BLLan AALan FMLan 图 5 部分菌株中类羊毛硫素合成酶结构域模式图 Figure 5 The model graph for domains of predicted lanthipeptide synthetases from some bacteria

: 2471 LILan S_TKc 7 STYKc S_TKc STYKc SCLan BSLan SPLan LDLan AALan FMLan LILan 6 BLLan C- LANC_like BSLan SPLan 2 2.

8 : 2471 LILan S_TKc 7 STYKc S_TKc STYKc SCLan BSLan SPLan LDLan AALan FMLan LILan 6 BLLan C- LANC_like BSLan SPLan 蛋白质三级结构预测 8 SWISS-MODEL 6 LANC_like STYKc 3 SCLan 图 6 部分菌株中羊毛硫素合成酶三级结构预测模式图 Figure 6 The model graph on tertiary structure of lanthipeptide synthetases from some bacteria BSLan 3 讨论 4 8 SCLan LDLan 8 8 α β β α LANC-like S_TKc STYKc 8 8 [22] 8 LANC-like 8

9 2472 微生物学通报 Microbiol. China 2016, Vol.43, No.11 [24-26] Iftime [2] Type IV lanthipeptide synthetase Type IV lanthipeptide synthetase 参考文献 [1] Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions[j]. Molecular Microbiology, 2005, 56(4): [2] Iftime D, Jasyk M, Kulik A, et al. Streptocollin, a type IV lanthipeptide produced by Streptomyces collinus Tü 365[J]. ChemBioChem, 2015, 16(18): [3] Zhang Q, Yu Y, Vélasquez JE, et al. Evolution of lanthipeptide synthetases[j]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(45): [4] Jungmann NA, van Herwerden EF, Hügelland M, et al. The supersized class III lanthipeptide stackepeptin displays motif multiplication in the core peptide[j]. ACS Chemical Biology, 2016, 11(1): [5] Xie LL, Miller LM, Chatterjee C, et al. Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity[j]. Science, 2004, 303(5658): [6] Marsh AJ, O Sullivan O, Ross RP, et al. In silico analysis highlights the frequency and diversity of type 1 lantibiotic gene clusters in genome sequenced bacteria[j]. BMC Genomics, 2010, 11: 679 [7] Begley M, Cotter PD, Hill C, et al. Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins[j]. Applied and Environmental Microbiology, 2009, 75(17): [8] Haft DH, Basu MK, Mitchell DA. Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family[j]. BMC Biology, 2010, 8: 70 [9] Bierbaum G, Sahl HG. Lantibiotics: mode of action, biosynthesis and bioengineering[j]. Current Pharmaceutical Biotechnology, 2009, 10(1): 2-18 [10] Guo SY, Li XY, He PF, et al. Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants[j]. Journal of Industrial Microbiology & Biotechnology, 2015, 42(6): [11] van Heel AJ, de Jong A, Montalbán-López M, et al. BAGEL3: automated identification of genes encoding bacteriocins and (non-) bactericidal posttranslationally modified peptides[j]. Nucleic Acids Research, 2013, 41: W448-W453 [12] Roy S, Maheshwari N, Chauhan R, et al. Structure prediction and functional characterization of secondary metabolite proteins of Ocimum[J]. Bioinformation, 2011, 6(8): [13] Petersen TN, Brunak S, von Heijne G, et al. SignalP 4.0: discriminating signal peptides from transmembrane regions[j]. Nature Methods, 2011, 8(10): [14] Huang HD, Lee TY, Tzeng SW, et al. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites[j]. Nucleic Acids Research, 2005, 33(Suppl 2): W226-W229 [15] McGuffin LJ, Bryson K, Jones DT. The PSIPRED protein structure prediction server[j]. Bioinformatics, 2000, 16(4): [16] Geourjon C, Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[j]. Computer Applications in the Biosciences: CABIOS, 1995, 11(6): [17] Letunic I, Doerks T, Bork P. SMART 7: recent updates to the protein domain annotation resource[j]. Nucleic Acids Research, 2012, 40: D302-D305 [18] Fink JL, Hamilton N. DomainDraw: a macromolecular feature drawing program[j]. In Silico Biology, 2007, 7(2): [19] Zhao DL, Li JY, Fang SM, et al. The bioinformatics analysis of Prp5 proteins in some model fungi[j]. Microbiology China, 2015, 42(8): (in Chinese),,,. Prp5 [J]., 2015, 42(8): [20] Bordoli L, Schwede T. Automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal[J]. Methods in Molecular Biology, 2012, 857: [21] Hsin J, Arkhipov A, Yin Y, et al. Unit 5.7, Chapter 5: Using VMD: An Introductory Tutorial[A]//Baxevanis AD. Current Protocols in Bioinformatics[M]. New York: John Wiley & Sons, 2008 [22] Goto Y, Ökesli A, van der Donk WA. Mechanistic studies of Ser/Thr dehydration catalyzed by a member of the LanL lanthionine synthetase Family[J]. Biochemistry, 2011, 50(5): [23] Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains[j]. Science, 1988, 241(4861): [24] Zhang YJ, Luo JC, Wu YP, et al. Research advancement of the biological control bacteria B. subtilis[j]. Life Science Instruments, 2009, 7(4): (in Chinese),,,. [J]., 2009, 7(4): [25] Li BQ, Lu XY, Guo QG, et al. Isolation and identification of lipopeptides and volatile compounds produced by Bacillus subtilis Strain BAB-1[J]. Scientia Agricultura Sinica, 2010, 43(17): (in Chinese),,,. BAB-1 [J]., 2010, 43(17): [26] Khochamit N, Siripornadulsil S, Sukon P, et al. Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain[j]. Microbiological Research, 2015, 170: 36-50

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇