粘质沙雷氏菌FS14全基因组测序分析及其Ⅵ型分泌系统翻译后调控蛋白和免疫蛋白的晶体结构

发布时间：2018-03-09 20:23

本文选题：沙雷氏菌　切入点：基因组　出处：《南京农业大学》2015年博士论文　论文类型：学位论文

【摘要】：粘质沙雷氏菌FS14是本实验室分离自白术根腐病害样品的一株产红色色素的肠杆菌。在平板对峙实验中,FS14表现出对植物病害真菌如尖孢镰刀菌和核盘菌等具有很强的拮抗作用,另外FS14对番茄病原细菌青枯雷尔氏菌也具有明显的抑制作用。虽然粘质沙雷氏菌的几丁质酶很早就被用于防治植物真菌病害,也有报道用粘质沙雷氏菌防治昆虫病害,但是对粘质沙雷氏菌拮抗植物病原的分子机制还不清楚。为了研究粘质沙雷氏菌FS14生物防治植物病害的机制,使其能稳定的用于农业生产,本研究对该菌株的全基因组进行了测序并做了比较基因组学分析,从基因水平了解沙雷氏菌的基本遗传特征,分析了拮抗相关基因,并表达和结晶了在FS14拮抗植物病原细菌中可能起关键作用的Ⅵ型分泌系统的翻译后调控蛋白和免疫蛋白。具体研究结果包括:1.本研究利用454焦磷酸测序对菌株FS14进行了全基因组测序。FS14的基因组由一个闭合环状染色体组成,基因组全长5,249,875 bp,G+C含量为59.46%(GenBank序列号:CP005927)。结合Glimmer和GeneMark预测得到4,761个编码序列,编码区域总长度为4,505,070bp,覆盖了FS14全基因组序列的85.8%。结合其他9株已公布的沙雷氏菌全基因组对该属进行了全面的比较基因组学分析,以FS14为参考菌株进行泛基因组分析,发现沙雷氏菌泛基因组的大小随着基因组数目的增加而增加,属于开放型泛基因组。10株沙雷氏菌的泛基因组含有10,837个编码基因,大约是单个基因组大小的2倍,表明沙雷氏菌不同菌株间存在丰富的遗传多样性;核心基因组包含1,964个编码基因(约是FS14基因数目的41%),其中大部分基因与细菌的基本功能相关;菌株特异性序列主要包括原噬菌体和假想蛋白,在这些特异性序列中还包括Ⅰ型限制修饰系统、Ⅲ型分泌系统、Ⅵ型分泌系统和碲抗性基因等,这些基因只出现在部分沙雷氏菌的基因组中。2.进一步分析发现,菌株FS14的基因组拥有多种与它的拮抗特性相关的基因,如灵菌红素、细菌素、嗜铁素、多重耐药性、表面活性剂、几丁质酶、蛋白酶、脂肪酶和磷脂酶等的编码基因。值得注意的是,在FS14的基因组中还发现了两个独立的Ⅵ型分泌系统(T6SS),其中一个与具有拮抗细菌功能的T6SS同源。用生物信息学方法找到已测序的沙雷氏菌的所有T6 S S,对其编码基因簇的组成结构进行比较分析,并利用130个T6SS保守蛋白ClpV的序列构建分子进化树,发现沙雷氏菌的T6SS分成四个分类群:T6SS-a,T6SS-b,T6SS-c和T6SS-d,其中T6SS-a保守存在于所有包含T6SS的沙雷氏菌中。菌株FS14的基因组包含T6SS-a和T6SS-b。这是目前为止,首次在沙雷氏菌的一个基因组中发现两种T6SS。3.沙雷氏菌Db10的T6SS-a被证明用于攻击同一环境中的其它细菌,因此菌株FS14的T6SS-a可能在其拮抗青枯雷尔氏菌中起关键作用。而T6SS的激活依赖丝氨酸/苏氦酸激酶(PpkA)和丝氨酸/苏氨酸磷酸酶(PppA)的翻译后调控。PpkA使T6SS中的Fha蛋白苏氨酸磷酸化,继而激活T6SS分泌复合物的组装和Hcp蛋白的分泌。PppA的作用与PpkA相反,使Fha去磷酸化。Fha是PpkA和PppA的共同作用底物,是T6S分泌装置的关键组分。翻译后调控使得T6SS能快速应对环境变化,对细菌适应环境并保持竞争优势起重要作用。将粘质沙雷氏菌T6SS-a的PpkA的N-端激酶结构域(PpkN)及C-端结构域(PpkAC)、PppA和Fha的基因通过PCR方法扩增并用大肠杆菌C43(DE3)表达,经亲和层析和凝胶过滤层析对重组蛋白进行分离纯化。体外表达并纯化的PpkAC有很强的降解DNA和RNA的核酸酶活性。纯化得到的PpkAN、PppA和Fha蛋白用坐滴气相扩散法进行蛋白质结晶条件的筛选和条件优化。PpkAN的初始结晶条件为:①0.1M NaCl,0.1M HEPES pH7.5,1.6M(NH4)2SO4;②0.1M HEPES-Na pH 7.5,1.5M Li2SO4;PppA的结晶条件为0.1M HEPES-Na pH7.5,0.75M Li2SO4;Fha蛋白较好的两个结晶条件为:①0.2M MgCl2,0.1M BIS-TRIS pH 5.5,25%PEG3350;②0.2M MgCl2,0.1M Tris-HCl,pH 8.5,30%PEG4000。通过对这些晶体进行初步X-光射线衍射分析发现这些晶体的分辨率并不理想。正在对这些结晶条件进一步优化,以便能够得到衍射能力良好的晶体并解析结构,为研究T6SS的翻译后调控机制提供结构基础。4.T6SS的效应蛋白被注射到缺少免疫蛋白的靶细胞中抑制靶细胞的生长,而相应的免疫蛋白能中和效应蛋白使自身细胞免受攻击。在FS14的T6SS-a中发现3个免疫蛋白的编码基因(L085_13755、L085_13760和L085_13765),其中L085_13755和L085_13760分别与粘质沙雷氏菌Db10的Rap2b和Rap2a有很高的同源性(90%),而L085_13765在菌株Db10中没有找到对应序列,但是在PDB中比对发现其与菌株Db10的T6SS免疫蛋白Rap2a具有32%的同源性,推测L085_13765编码FS14 T6SS-a的第三个免疫蛋白,命名为Rap2c。本研究用大肠杆菌C43(DE3)诱导表达Rap2c蛋白,经亲和层析和凝胶过滤层析对重组蛋白进行分离纯化,得到了高纯度且聚合状态单一的目的蛋白。将纯化得到的Rap2c蛋白浓缩至适当浓度后,用坐滴气相扩散法进行蛋白质结晶条件的筛选和条件优化。对在这些条件下生长得到的晶体进行初步X-光射线衍射分析发现这些晶体的分辨率约为7A。通过对Rap2c的二级结构和三级结构的预测,发现Rap2c同Tsi家族的免疫蛋白一样由5个α螺旋(α1-α5)折叠成束状物,Cys31-Cys95可以形成二硫键将α2和α4连接,α3螺旋可能是结合效应蛋白的关键部位,而α3-α4 loop可能在阻断效应蛋白的活性时起主要作用。
[Abstract]:Serratia marcescens FS14 isolated from Atractylodes root rot disease samples of a strain producing red pigment of Enterobacteriaceae in our laboratory. In plate confrontation test, FS14 has shown strong antagonism against plant disease fungi such as Fusarium oxysporum and Sclerotinia sclerotiorum, in addition FS14 on tomato pathogen Ralstonia ray Yersinia also has obvious inhibitory effects. Although Serratia marcescens chitinase has long been used for prevention and treatment of fungal diseases of plants, have also been reported with Serratia marcescens insect disease, but the molecular mechanism of Serratia marcescens against plant pathogens is not clear. In order to study the mechanism of Serratia marcescens FS14 biological control of plant diseases, which can be used to stabilize the agricultural production, the Research on the whole genome of the strain was sequenced and the comparative genomics analysis, from gene level to understand the basic genetics of Serratia Transmission characteristics, analysis of the antagonistic related genes, and the expression and regulation of protein crystallization and immune protein of type VI may play a key role in FS14 against plant pathogenic bacteria in the secretory system of translation. The research results include: 1. the study of the strain FS14 were whole genome sequencing the genome of.FS14 consists of a closed ring chromosome using 454 pyrosequencing, full-length 5249875 BP, the content of G+C is 59.46% (GenBank serial number: CP005927). The combination of Glimmer and GeneMark predicted 4761 encoding sequence, the total length of encoding region was 4505070bp, covering the whole genome sequence of FS14 85.8%. with other 9 strains have been published on the whole genome of Serratia sp. a comprehensive study of comparative genomic analysis, using FS14 as the reference strains of Pan genome analysis, found that Serratia pan genome size with genome number Increases, belong to the open type pan genome of.10 strain Shaleishi bacteria pan genome contains 10837 genes encoding, is about 2 times that of a single genome size, showed that Serratia marcescens isolates existed abundant genetic diversity; the core genome contains 1964 encoding gene (FS14 gene number is about 41%). One of the most basic functions of the related gene and bacterial strains; specific sequences including the original phage and hypothetical protein, also included in these specific sequences of type I restriction modification system, type III secretion system, the type VI secretion system and tellurium resistance genes, these genes appear only in part of the genome of Serratia.2. further analysis showed that the genome of strain FS14 has a variety of characteristics associated with its antagonistic genes, such as prodigiosin, bacteriocin, siderophore, multidrug resistance, surfactant, chitinase, The gene encoding protease, lipase and phospholipase etc.. It is worth noting that in the genome of FS14 was found in two independent type VI secretion system (T6SS), which has a homology with antagonistic bacteria T6SS. Sarre's function of bacteria by Bioinformatics Method to find sequenced all T6 S S and compare the composition of its encoding gene cluster, and constructed a phylogenetic tree using a sequence of 130 conserved T6SS protein ClpV, found that Serratia T6SS divided into four taxa: T6SS-a, T6SS-b, T6SS-c and T6SS-d, the conservative T6SS-a exists in all T6SS containing Serratia strain FS14. The genome contains T6SS-a and T6SS-b. which is now in a genome of Serratia found two T6SS.3. Serratia Db10 T6SS-a proved for other bacterial attack in the same environment for the first time, so strain FS14 T6S S-a may play a key role in the antagonism of Ralstonia solanacearum. Activation of T6SS dependent serine / threonine kinase (PpkA) and helium acid serine / threonine phosphatase (PppA) post-translational regulation of.PpkA Fha protein threonine phosphorylation of T6SS, PpkA and.PppA secretion function and activation of Hcp protein and assembly the secretion of T6SS complexes of dephosphorylated Fha on the contrary,.Fha is the combined effect of PpkA and PppA substrate, T6S group is the key device. The secretion of post-translational regulation makes T6SS can quickly respond to environmental changes, to adapt to the environment of bacteria and maintain the competitive advantage. It will play an important role in Serratia marcescens T6SS-a PpkA the N- terminal kinase domain (PpkN) and C- domain (PpkAC), PppA and Fha genes were amplified by PCR and Escherichia coli C43 (DE3) expression of recombinant protein was purified by affinity chromatography and gel filtration chromatography in vitro expression. Nuclease activity and purification of PpkAC has a strong degradation of DNA and RNA. The purified PpkAN, initial crystallization condition screening and optimization of.PpkAN PppA and Fha protein using the sitting drop vapor diffusion method for protein crystallization conditions as follows: 0.1M NaCl, 0.1M HEPES pH7.5,1.6M (NH4) 2SO4; 0.1M HEPES-Na pH 7.5,1.5M Li2SO4; PppA 0.1M HEPES-Na as the crystallization conditions of pH7.5,0.75M Li2SO4; two Fha protein crystallization conditions better: 1 0.2M MgCl2,0.1M BIS-TRIS pH 5.5,25%PEG3350; MgCl2,0.1M Tris-HCl pH 8.5,30%PEG4000. 0.2M, through a preliminary X- ray diffraction analysis shows that these crystals are not ideal for the resolution of these crystals. These are further optimization of crystallization conditions and in order to be able to get good crystal diffraction ability and analytical structure, provide the structural basis of.4.T6SS regulation effect and mechanism of T6SS after translation The protein was injected into the target cells lack immune protein in inhibiting cell growth, while the corresponding immune protein can neutralize effector proteins to make their own cells from attack. The genes encoding the 3 immune protein found in FS14 T6SS-a (L085_13755, L085_13760 and L085_13765), the L085_13755 and L085_13760 respectively with Serratia marcescens Db10 Rap2b and Rap2a have very high homology (90%), and L085_13765 in strain Db10 was not found in the corresponding sequence, but found that the strain Db10 and T6SS immune protein Rap2a has 32% homology in the PDB ratio of third, suggesting that L085_13765 encoding FS14 T6SS-a protein, named Rap2c. in this study Escherichia coli C43 (DE3) induced the expression of Rap2c protein, the recombinant protein was purified by affinity chromatography and gel filtration chromatography, obtained high purity and aggregation of single target protein will be pure. The obtained Rap2c protein concentration to the appropriate concentration, screening and optimization of protein crystallization conditions using the sitting drop vapor diffusion method. A preliminary X- ray diffraction analysis showed that the crystal resolution is about 7A. by predicting the two level structure of Rap2c and the three stage structure on the growth obtained in these conditions crystal, found that the immune protein Rap2c with Tsi as the family consists of 5 alpha helix (alpha 1- alpha 5) folded into clusters, Cys31-Cys95 can form two disulfide bonds in the alpha 2 and alpha 4 alpha 3 screw connection, may be the key site binding protein, alpha 3- alpha 4 loop may play a major role in effect of blocking the activity of the protein.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S432.4

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