本文選題：內(nèi)源性隱秘質(zhì)粒pMF1 + 主動分配系統(tǒng)��； 參考：《山東大學》2016年博士論文

【摘要】：細菌質(zhì)粒,在1952年由Joshua Lederberg發(fā)現(xiàn),是一類獨立于染色體外,能自主復制的遺傳因子。質(zhì)�；蚪M一般包括一系列必需基因,比如負責復制、分配等維持遺傳穩(wěn)定的基因,還包括各種各樣的附屬基因。質(zhì)粒對于細菌的進化適應具有重要作用,一是由于其能在不同的遺傳距離較遠的宿主之間轉(zhuǎn)移,通過重組、轉(zhuǎn)座等實現(xiàn)基因交流；二是其能編碼很多對細菌有利的生態(tài)學表型,如抗生素、毒素、重金屬抗性等。然而,自然界中還存在大量的隱秘質(zhì)粒,這些質(zhì)粒并不攜帶明顯的宿主表型優(yōu)勢基因,而且,大范圍篩選證實大約一半的質(zhì)粒不具有可移動性。質(zhì)粒存在會給宿主帶來代謝負擔,質(zhì)粒DNA的維持和修復以及質(zhì)粒蛋白的合成會消耗宿主細胞的原料,占據(jù)細胞的器官,如核糖體,破壞細胞的內(nèi)環(huán)境。所以質(zhì)粒存在的前提是對質(zhì)粒上有益附屬性狀的陽性篩選超過質(zhì)粒帶給宿主的負擔,但是,對有益性狀的持續(xù)選擇最終會使這些基因被整合到宿主染色體上。許多長時間細菌-質(zhì)粒共培養(yǎng)的實驗進化研究表明盡管會給宿主帶來生理學負擔,即使是在沒有正選擇的情況下,質(zhì)粒也不會很容易的從細菌種群中丟失。目前關于質(zhì)粒保存機制有多種解釋,比如位點特異性重組、翻譯后自殺系統(tǒng)、低拷貝質(zhì)粒的主動分配系統(tǒng)、接合質(zhì)粒的高接合率、非移動性質(zhì)粒的陽性選擇和代償性適應等等。但是,隱秘質(zhì)粒在細菌宿主內(nèi)的保存機制及其給宿主帶來的影響仍然不清楚。粘細菌是一類特殊的細菌類群,具有復雜的細胞間協(xié)同行為和龐大的基因組。粘細菌基因組的典型特點是存在大量的基因復制和水平基因轉(zhuǎn)移現(xiàn)象,比如Sorangium cellulosum So0157-2(14.78Mb)的基因組中近40%的基因可能來自水平轉(zhuǎn)移,暗示了粘細菌基因組易于整合外源DNA并進行染色體自我重組。整合外源DNA需要移動工具,比如質(zhì)粒、噬菌體,但是,與其明顯的基因組擴張相反的是,在粘細菌中并未發(fā)現(xiàn)普遍的質(zhì)粒存在現(xiàn)象。pMF1,來自于Myxococcusfulvus 124B02,是目前發(fā)現(xiàn)的唯一能在粘細菌細胞中自主復制的內(nèi)源質(zhì)粒。pMF1對于研究為何M.fulvus 124B02能夠包含內(nèi)源質(zhì)粒、質(zhì)粒給M.fulvus124B02宿主帶來的影響以及粘細菌基因組的進化具有重要意義。論文圍繞粘細菌內(nèi)源質(zhì)粒pMF1的存在機制展開,主要研究內(nèi)容與研究結(jié)果如下：1、pMF1復制和分配遺傳穩(wěn)定區(qū)域功能模式分析。采用PEG6000沉淀法提取了M. xanthus DZ1 pZJY41的復制中間體,確定了pMF1的復制方式為theta型,這種方式是大部分革蘭氏陰性細菌中質(zhì)粒的復制方式。但是,與經(jīng)典的repABC質(zhì)粒的復制和分配方式不同,pMF1的復制和分配功能是由兩個單獨的操縱子負責(pMF1.13-pMF1.16, pMF1.21-pMF1.23),基因結(jié)構、調(diào)控網(wǎng)絡更復雜,我們對維持低拷貝質(zhì)粒穩(wěn)定存在的主動分配系統(tǒng)進行了更為深入的研究。pMF1質(zhì)粒的par loci除了包含其他低拷貝質(zhì)粒都含有的編碼ATPase (pMF1.22, parA)、DNA-binding protein (pMF1.23, parB)的基因以及parS位點以外,還包含一個額外的基因(pMFl.21),我們命名為parC。這與其他低拷貝質(zhì)粒的主動分配系統(tǒng)有明顯不同,暗示了pMF1質(zhì)粒在完成復制進行分配時采用了一種新穎的方式。在論文的第二部分,我們對該基因進行了研究。parC位于promoter和parA之間,并且與parA在序列上有4個堿基的重疊。這種序列上的組合方式暗示了parC可能具有某種功能。將parC進行全基因敲除后,重組質(zhì)粒的穩(wěn)定性下降到與pZJY41相似,對粘球菌宿主M.xanthus DZ1最大生長量的影響也顯著下降,表明parC參與了par loci精確分配質(zhì)粒和影響宿主生長的過程。融合熒光報告基因結(jié)果顯示parC在粘球菌宿主中能正常表達成蛋白,是以蛋白質(zhì)的形式發(fā)揮作用。通過與數(shù)據(jù)庫進行比對并沒有找到ParC在序列和結(jié)構上的同源蛋白。對其二級結(jié)構進行預測發(fā)現(xiàn)ParC含有大量的a螺旋,大約80%的氨基酸都形成了a螺旋。同源模建結(jié)果發(fā)現(xiàn)ParC形成一個發(fā)卡樣的長螺旋,該長螺旋逆時針旋轉(zhuǎn)成一個類似DNA超螺旋結(jié)構的右手螺旋。表面電勢分析顯示在長螺旋的頂端(N-端)廣泛分布著一些帶正電荷的氨基酸,而底部(C-端)則富含帶負電荷的氨基酸。結(jié)合ParC形成三聚體的實驗結(jié)果,我們可以總結(jié)出ParC螺旋利用半胱氨酸形成二硫鍵,組裝成3個螺旋貼在一起的N-端帶正電,C-端帶負電的“棒狀”結(jié)構。pMF1的DNA-binding protein ParB是一個堿性蛋白,帶正電荷,而細胞內(nèi)的DNA是帶負電荷的,ParC這種電荷的不均勻分布是否是為了與這兩者相互作用呢?實驗表明ParC確實能增強ParB與ItA(parS位點)的結(jié)合作用,但其本身與ItA并不結(jié)合。而且ParC與ori(10953-13980)及par loci (17242-50)區(qū)均沒有結(jié)合作用。體內(nèi)和體外實驗表明ParC與ParB之間也沒有相互作用。在低拷貝質(zhì)粒的分配過程中,第一步便是大量的ParB蛋白與parS結(jié)合,形成分配復合物,而我們的結(jié)果表明ParC不參與質(zhì)粒分配的第一步。pMF1質(zhì)粒的復制和分配方式不同于其他低拷貝質(zhì)粒,對其機制我們在做進一步研究。對于隱秘質(zhì)粒來說,僅有完整的復制和分配功能不能保證其在宿主漫長進化過程中的穩(wěn)定存在。接下來我們將研究目標擴展到整個質(zhì)粒和宿主,從基因組學的角度研究質(zhì)粒-宿主的進化歷史。2、pMF1質(zhì)粒和宿主M.fulvus 124B02基因組組學研究暗示了兩者的共進化。我們對pMF1質(zhì)粒上的23個基因所編碼的蛋白分別進行功能來源預測,并歸為四類。其中14個蛋白與粘細菌密切相關,質(zhì)粒上約1/3(8個)的編碼蛋白只能與Mstipitatus DSM14675比對到同源蛋白,另外,1個來自于Stigmatella aurantiaca,1個來自于Anaeromyxobacter,1個來自于Chondromyces crocatus和Sorangium cellulosum,3個來自于粘細菌眾多種屬。9個pMF1蛋白在數(shù)據(jù)庫中比對不到任何的同源蛋白,屬于pMF1特有。但是,很多蛋白的功能仍然未知。轉(zhuǎn)錄組數(shù)據(jù)表明在23個基因中,轉(zhuǎn)錄水平最高的是pMF1.17,pMF1.18,其次是pMF1.12。鏈特異性轉(zhuǎn)錄組和RT-PCR結(jié)果表明pMF1包含6個操縱子,占全部基因比例的87%(20/23)。接下來我們對宿主M. fulvus 124B02進行了基因組全測序,結(jié)果表明M. fulvus 124B02包含一個環(huán)形染色體,大小為11,048,835 bp,以及一個環(huán)形質(zhì)粒,也就是pMF1。染色體和質(zhì)�；蚪M的GC含量相似,分別為69.96%和68.7%。全基因組進化樹和共線性比對表明M.fulvus 124B02與M.stipitatus DSM14675同源性最高,二者在基因組大小上也最接近。與其他粘球菌相比,M.fulvus 124B02的基因組有1-2 Mb的擴張,但其在直系同源和旁系同源基因比例上并沒有明顯差異。限制修飾系統(tǒng)和CRISPR-Cas系統(tǒng)比較分析發(fā)現(xiàn),M.fulvus 124B02的防御系統(tǒng)更加薄弱,其Cas蛋白操縱子比其他粘球菌要少1/2-2/3,記錄外源DNA的spacers也少于其他同種或同屬的粘細菌,限制修飾系統(tǒng)類型和修飾酶種類也較少。同源比對結(jié)果顯示pMF1上的某些基因來自于其他粘細菌,暗示了pMF1曾經(jīng)在不同粘細菌之間水平轉(zhuǎn)移,而且與M.stipitatus DSM14675的同源基因最多。粘球菌屬產(chǎn)生于47-51百萬年前,而M.fulvus 124B02與M.stipitatus DSM14675在大約41百萬年前時由共同祖先分化而來,相對薄弱的免疫系統(tǒng)解釋了為什么pMF1最終在M.fulvus 124B02中保存下來,并與M.fulvus 124B02共同進化,穩(wěn)定存在。3、pMFl在宿主M.fulvus 124B02中發(fā)揮維持宿主基因組穩(wěn)定的作用為了探明pMF1穩(wěn)定存在于M.fulvus 124B02中的機制,我們構建了質(zhì)粒消除菌株,在實驗室條件下模擬pMF1與宿主M.fulvus 124B02的進化。利用質(zhì)粒不相容原理可以將pMF1自M.fulvus 124B02中消除,而且pMF1的消除沒有顯著影響宿主的生長、運動、發(fā)育等表型,說明pMF1對宿主的影響并不是短時間的表型影響。在進行實驗室傳代時,我們設定了三種培養(yǎng)條件,分別以豐富的CYE、貧瘠的dead cells和捕食性的living cells為食物來源。結(jié)果發(fā)現(xiàn)只有在以貧瘠的dead cells為營養(yǎng)時,pMF1能穩(wěn)定存在,而在其他兩種條件下傳代的菌株中,pMF1在7-8周時便檢測不到。為了找到影響pMF1穩(wěn)定存在的相關基因,我們對三種條件傳代的菌株進行測序,對篩選出的基因進行敲除,重復傳代實驗,最終確定了可能相關的一些基因。我們篩選到了pMF1穩(wěn)定存在的實驗室條件,在該條件下,對經(jīng)過較長時間共進化的菌株進行表型分析時發(fā)現(xiàn)不攜帶質(zhì)粒的菌株其發(fā)育能力下降程度要明顯高于攜帶質(zhì)粒的菌株,CYE平板上的124B02/free菌株甚至由聚團生長變成分散生長,暗示了pMF1對宿主的影響可能是經(jīng)過長時間的逐漸積累。為了驗證這一猜想,我們對進化菌株進行基因組測序,結(jié)果表明124B02/free菌株的基因組突變率要明顯高于124B02/4111和124B02/pMF1菌株,pMF1或者pZJY4111質(zhì)粒的存在能降低基因組突變率,而且這些突變的發(fā)生是隨機的,沒有基因組位置和基因功能的偏好性。pMF1和pZJY4111的共同部分是質(zhì)粒的ori和par loci,而pZJY4111質(zhì)粒在進化傳代過程中ori被宿主剪切,只剩下par loci的現(xiàn)象表明可能是par loci發(fā)揮了穩(wěn)定基因組,防止基因組發(fā)生突變的作用。綜上所述,pMF1的發(fā)現(xiàn),不僅成功解決了粘細菌的遺傳操作問題,同時為我們了解粘細菌基因組進化提供了指導。據(jù)此,我們推測了pMF1隱秘質(zhì)粒穩(wěn)定存在的機制模型。在粘細菌的進化歷史過程中,做為基因移動的載體,pMF1曾經(jīng)在粘細菌之間水平轉(zhuǎn)移。由于結(jié)構簡單、拷貝數(shù)高,質(zhì)粒被認為是快速的基因進化器,可以加速宿主基因組的進化。由于具有相對較弱的免疫防御系統(tǒng),pMF1更容易進入M.fulvus 124B02宿主細胞內(nèi)。pMF1通過參與宿主基因組的錯配修復過程,最終被M.fulvus 124B02需要而保存下來。
[Abstract]:Bacterial plasmids, discovered in 1952 by Joshua Lederberg, are a class of genetic factors independent of dyed in vitro and independently replicating. Plasmid genomes generally include a series of essential genes, such as genes responsible for replication, distribution, and other genes that maintain genetic stability, and include a variety of attached genes. Plasmids are important for the evolutionary adaptation of bacteria. The function, one is that it can transfer between different genetic distance hosts, through recombination and transposing, and so on. Two, it can encode many ecological phenotypes beneficial to bacteria, such as antibiotics, toxins, heavy metal resistance and so on. However, there are a large number of cryptic plasmids in nature, which do not carry the obvious plasmid. The host phenotype predominance gene, and the large range screening confirms that about half of the plasmids do not have mobility. Plasmids present a metabolic burden on the host, the maintenance and repair of plasmid DNA, and the synthesis of plasmid proteins will consume the host cell's raw materials, occupy the cell organs, such as ribosomes, and destroy the inner environment of the cells. So plasmids The precondition is that the positive screening of beneficial ancillary traits on the plasmid exceeds the burden of the plasmid to the host, but the continuous selection of beneficial traits will eventually integrate these genes into the host chromosome. In the absence of positive selection, plasmids will not be easily lost from bacterial populations. There are many interpretations of plasmid preservation mechanisms, such as site specific recombination, posttranslational suicide system, active distribution system of low copy plasmids, high conjugation rate of conjugative plasmids, positive selection of non mobility plasmids and compensatory adaptation. However, the preservation mechanism of the cryptic plasmid in the bacterial host and its effects on the host are still unclear. The bacteria are a special group of bacteria, with complex intercellular coordination and a huge genome. The typical characteristics of the bacterial genome are the existence of a large number of gene replicas and horizontal gene transfer phenomena. Nearly 40% of the genes in the genome of Sorangium cellulosum So0157-2 (14.78Mb) may come from horizontal metastasis, suggesting that the bacterial genome is easy to integrate exogenous DNA and recombine chromosomes. Integration of exogenous DNA requires mobile tools, such as plasmids, phages, but, contrary to the obvious genomic dilatation, is in the bacteria. No common plasmid presence.PMF1, from Myxococcusfulvus 124B02, is the only endogenous plasmid.PMF1 that can be independently replicated in the bacterial cells of the bacterial cells. The effect of M.fulvus 124B02 on the inclusion of endogenous plasmids, the effects of plasmids on the M.fulvus124B02 host and the evolution of the bacterial genome of the clay bacteria The main contents and research results are as follows: 1, 1, pMF1 replication and distribution of genetic stability regional functional model analysis. The replication intermediate of M. xanthus DZ1 pZJY41 is extracted by PEG6000 precipitation method, and the replication mode of pMF1 is theta type, this way is Most Gram-negative bacteria are replicating the plasmid. However, unlike the classical repABC plasmid replication and distribution, the replication and distribution function of pMF1 is responsible for two individual operon (pMF1.13-pMF1.16, pMF1.21-pMF1.23), gene structure, and the regulatory network is more complex, and we are the master of maintaining the stability of low copy plasmids. The dynamic distribution system carried out a more in-depth study of the.PMF1 plasmid par loci in addition to the encoded ATPase (pMF1.22, parA), the DNA-binding protein (pMF1.23, parB) gene and the parS site, including the other low copy plasmids, and the parS loci, which also included an additional gene (pMFl.21), which we named it with other low copy plasmids. The active distribution system is distinctly different, suggesting a novel approach to the allocation of pMF1 plasmids in the completion of replication. In the second part of the paper, we have studied the gene between the.ParC and the parA, and there are 4 bases in the sequence with parA. The combination of this sequence suggests that parC is available. After total gene knockout of parC, the stability of the recombinant plasmid decreased to the same as pZJY41, and the effect on the maximum growth of M.xanthus DZ1 was also significantly decreased, indicating that parC participated in the par loci accurate allocation of plasmids and the process of influencing the growth of the host. Fusion fluorescent reporter gene results showed parC in sticky ball. The bacteria in the host can be expressed as proteins in the form of protein. By comparison with the database, the ParC is not found in the sequence and structure of homologous proteins. The prediction of the secondary structure shows that ParC contains a large number of a helices and about 80% of the amino acids form a a spiral. Homologous modeling results found the formation of ParC A long helix like a long spiral, which rotates into a right-handed spiral similar to a DNA super spiral structure. Surface potential analysis shows that some positive charged amino acids are widely distributed at the top of the long helix (N- end), while the bottom (C- end) is rich in negatively charged amino acids. Experimental results of a trimer formed by combining ParC with ParC It can be concluded that ParC helix uses cysteine to form two sulfur bonds, assembled into 3 helically attached N- ends with positive electricity, and the DNA-binding protein ParB of the "rod like" structure.PMF1 with negative electricity in the C- end is an alkaline protein with positive charge, and the DNA in the cell is negatively charged, and the uneven distribution of ParC, a charge, is for the purpose of The interaction between the two? The experiment shows that ParC does enhance the binding of ParB to ItA (parS site), but it does not bind to ItA itself. And ParC has no binding to ori (10953-13980) and par loci (17242-50) regions. In vivo and in vitro experiments show that ParC and ParB are not interacting. In the course, the first step is to combine a large number of ParB proteins with parS to form a distribution complex, and our results show that the first step of.PMF1 plasmids that ParC does not participate in plasmids is different from other low copy plasmids, and we are doing further research on its mechanism. For the cryptic plasmid, only complete replication and distribution are available. Function does not guarantee its stable existence in the long evolution process of the host. Next, we extend the research objectives to the whole plasmid and host, and study the evolution history of plasmid host from the genomics perspective.2, pMF1 plasmids and host M.fulvus 124B02 genomic studies suggest the co evolution of both. We have 23 on the pMF1 plasmid. The proteins encoded by the genes were predicted for functional sources and were classified as four. 14 proteins were closely related to the bacteria, and the encoding proteins on the plasmid 1/3 (8) were only compared with Mstipitatus DSM14675 to the homologous protein, and 1 were from Stigmatella aurantiaca, 1 from Anaeromyxobacter, and 1 from Chondromyc. Es crocatus and Sorangium cellulosum, 3 from a variety of.9 pMF1 proteins from the bacteria of the bacteria, are specific to no homologous protein in the database and are specific to pMF1. However, the functions of many proteins are still unknown. The transcriptional data indicate that in the 23 genes, the highest level of transcription is pMF1.17, pMF1.18, and next to pMF1.12. chain specificity The transcriptome and RT-PCR results showed that pMF1 contained 6 operon, accounting for 87% (20/23) of the total gene proportion. Then we sequenced the genome of the host M. fulvus 124B02. The results showed that M. fulvus 124B02 contained a ring chromosome, the size of 11048835 BP, and a ring plasmid, that is, pMF1. chromosome and plasmid gene. The GC content of the group was similar. The 69.96% and 68.7%. whole genome evolution trees and the co linear alignment showed that M.fulvus 124B02 and M.stipitatus DSM14675 had the highest homology, and the two were the closest in the size of the genome. The genome of M.fulvus 124B02 had a 1-2 Mb expansion compared with other visco coccus, but it was in the direct and accessory homologous genes. There was no significant difference in the proportion of the modified system and the CRISPR-Cas system. It was found that the defense system of M.fulvus 124B02 was weaker, the Cas protein operon was less 1/2-2/3 than the other viscos, and the spacers of the foreign DNA was less than that of the other homohomologous or congeneric strains of Micrococcus, and the types of modified systems and the types of modified enzymes were also limited. The homologous alignment results showed that some of the genes on pMF1 were derived from other bacteria, suggesting that pMF1 had shifted horizontally between different bacteria and was the most homologous to M.stipitatus DSM14675. The genus neococcus was produced 47-51 million years ago, and M.fulvus 124B02 and M.stipitatus DSM14675 were shared about 41 million years ago. An ancestral differentiation, the relatively weak immune system explains why pMF1 is eventually preserved in M.fulvus 124B02 and coevolved with M.fulvus 124B02, stable in.3, and pMFl plays the role of maintaining host genome stability in host M.fulvus 124B02 in order to identify the mechanism that pMF1 is stable in M.fulvus 124B02. Plasmid elimination strains were built to simulate the evolution of pMF1 and host M.fulvus 124B02 under laboratory conditions. Using plasmid incompatibility principle can eliminate pMF1 from M.fulvus 124B02, and the elimination of pMF1 has not significantly affected the host growth, movement, development and other phenotypes, indicating that pMF1's effect on the host is not a short time phenotypic effect. During the laboratory passage, we set three culture conditions with rich CYE, poor dead cells and predatory living cells as food sources. The results showed that pMF1 could be stable only when poor dead cells was used as nutrition, and pMF1 was not detected at 7-8 weeks in the other two conditions. In order to find the related genes that affect the stable existence of pMF1, we sequenced the strains of three conditional passages, knocked out the selected genes, repeated the generation experiments, and finally identified some genes that might be related. We screened the stable laboratory conditions of pMF1, and in this condition, we have coevolved for a long time. In phenotypic analysis, it was found that the growth ability of the strain which did not carry the plasmid was significantly higher than that of the plasmid carrying strain. The 124B02/free strain on the CYE plate even grew from the cluster to the dispersed growth, suggesting that the effect of pMF1 on the host may be accumulated for a long time. In order to verify this conjecture, we are right Genomic sequencing of evolutionary strains showed that the mutation rate of 124B02/free strains was significantly higher than that of 124B02/4111 and 124B02/pMF1 strains. The presence of pMF1 or pZJY4111 plasmids could reduce the mutation rate of the genome, and the occurrence of these mutations was random, without the preference of genomic location and gene function of.PMF1 and pZJY4111. The common part is the ori and par loci of the plasmid, while the pZJY4111 plasmid is cut by the host during the evolutionary passage, only the par loci is left to show that par loci exerts a stable genome to prevent the mutation of the genome. In summary, the discovery of pMF1 not only successfully solved the genetic manipulation of the bacteria, but also made a successful solution to the genetic manipulation of the bacteria. We understand the genome evolution of the bacterial genome. According to this, we speculated the mechanism model for the stability of the pMF1 cryptic plasmid. In the evolutionary history of the bacteria, the plasmid was used as a carrier of gene movement, and pMF1 had been transferred between the bacteria. The plasmid was considered to be a fast gene evolutional because of its simple structure and the high number of custle shells. It can accelerate the evolution of the host genome. Because of the relatively weak immune defense system, pMF1 is more likely to enter the M.fulvus 124B02 host cell.PMF1 by participating in the mismatch repair process of the host genome, and is eventually saved by M.fulvus 124B02.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q78
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本文編號：1884189