粘细菌内源性隐秘质粒pMF1存在机制研究

发布时间：2018-05-13 17:49

  本文选题：内源性隐秘质粒pMF1 + 主动分配系统　； 参考：《山东大学》2016年博士论文

【摘要】：细菌质粒,在1952年由Joshua Lederberg发现,是一类独立于染色体外,能自主复制的遗传因子。质粒基因组一般包括一系列必需基因,比如负责复制、分配等维持遗传稳定的基因,还包括各种各样的附属基因。质粒对于细菌的进化适应具有重要作用,一是由于其能在不同的遗传距离较远的宿主之间转移,通过重组、转座等实现基因交流；二是其能编码很多对细菌有利的生态学表型,如抗生素、毒素、重金属抗性等。然而,自然界中还存在大量的隐秘质粒,这些质粒并不携带明显的宿主表型优势基因,而且,大范围筛选证实大约一半的质粒不具有可移动性。质粒存在会给宿主带来代谢负担,质粒DNA的维持和修复以及质粒蛋白的合成会消耗宿主细胞的原料,占据细胞的器官,如核糖体,破坏细胞的内环境。所以质粒存在的前提是对质粒上有益附属性状的阳性筛选超过质粒带给宿主的负担,但是,对有益性状的持续选择最终会使这些基因被整合到宿主染色体上。许多长时间细菌-质粒共培养的实验进化研究表明尽管会给宿主带来生理学负担,即使是在没有正选择的情况下,质粒也不会很容易的从细菌种群中丢失。目前关于质粒保存机制有多种解释,比如位点特异性重组、翻译后自杀系统、低拷贝质粒的主动分配系统、接合质粒的高接合率、非移动性质粒的阳性选择和代偿性适应等等。但是,隐秘质粒在细菌宿主内的保存机制及其给宿主带来的影响仍然不清楚。粘细菌是一类特殊的细菌类群,具有复杂的细胞间协同行为和庞大的基因组。粘细菌基因组的典型特点是存在大量的基因复制和水平基因转移现象,比如Sorangium cellulosum So0157-2(14.78Mb)的基因组中近40%的基因可能来自水平转移,暗示了粘细菌基因组易于整合外源DNA并进行染色体自我重组。整合外源DNA需要移动工具,比如质粒、噬菌体,但是,与其明显的基因组扩张相反的是,在粘细菌中并未发现普遍的质粒存在现象。pMF1,来自于Myxococcusfulvus 124B02,是目前发现的唯一能在粘细菌细胞中自主复制的内源质粒。pMF1对于研究为何M.fulvus 124B02能够包含内源质粒、质粒给M.fulvus124B02宿主带来的影响以及粘细菌基因组的进化具有重要意义。论文围绕粘细菌内源质粒pMF1的存在机制展开,主要研究内容与研究结果如下：1、pMF1复制和分配遗传稳定区域功能模式分析。采用PEG6000沉淀法提取了M. xanthus DZ1 pZJY41的复制中间体,确定了pMF1的复制方式为theta型,这种方式是大部分革兰氏阴性细菌中质粒的复制方式。但是,与经典的repABC质粒的复制和分配方式不同,pMF1的复制和分配功能是由两个单独的操纵子负责(pMF1.13-pMF1.16, pMF1.21-pMF1.23),基因结构、调控网络更复杂,我们对维持低拷贝质粒稳定存在的主动分配系统进行了更为深入的研究。pMF1质粒的par loci除了包含其他低拷贝质粒都含有的编码ATPase (pMF1.22, parA)、DNA-binding protein (pMF1.23, parB)的基因以及parS位点以外,还包含一个额外的基因(pMFl.21),我们命名为parC。这与其他低拷贝质粒的主动分配系统有明显不同,暗示了pMF1质粒在完成复制进行分配时采用了一种新颖的方式。在论文的第二部分,我们对该基因进行了研究。parC位于promoter和parA之间,并且与parA在序列上有4个碱基的重叠。这种序列上的组合方式暗示了parC可能具有某种功能。将parC进行全基因敲除后,重组质粒的稳定性下降到与pZJY41相似,对粘球菌宿主M.xanthus DZ1最大生长量的影响也显著下降,表明parC参与了par loci精确分配质粒和影响宿主生长的过程。融合荧光报告基因结果显示parC在粘球菌宿主中能正常表达成蛋白,是以蛋白质的形式发挥作用。通过与数据库进行比对并没有找到ParC在序列和结构上的同源蛋白。对其二级结构进行预测发现ParC含有大量的a螺旋,大约80%的氨基酸都形成了a螺旋。同源模建结果发现ParC形成一个发卡样的长螺旋,该长螺旋逆时针旋转成一个类似DNA超螺旋结构的右手螺旋。表面电势分析显示在长螺旋的顶端(N-端)广泛分布着一些带正电荷的氨基酸,而底部(C-端)则富含带负电荷的氨基酸。结合ParC形成三聚体的实验结果,我们可以总结出ParC螺旋利用半胱氨酸形成二硫键,组装成3个螺旋贴在一起的N-端带正电,C-端带负电的“棒状”结构。pMF1的DNA-binding protein ParB是一个碱性蛋白,带正电荷,而细胞内的DNA是带负电荷的,ParC这种电荷的不均匀分布是否是为了与这两者相互作用呢?实验表明ParC确实能增强ParB与ItA(parS位点)的结合作用,但其本身与ItA并不结合。而且ParC与ori(10953-13980)及par loci (17242-50)区均没有结合作用。体内和体外实验表明ParC与ParB之间也没有相互作用。在低拷贝质粒的分配过程中,第一步便是大量的ParB蛋白与parS结合,形成分配复合物,而我们的结果表明ParC不参与质粒分配的第一步。pMF1质粒的复制和分配方式不同于其他低拷贝质粒,对其机制我们在做进一步研究。对于隐秘质粒来说,仅有完整的复制和分配功能不能保证其在宿主漫长进化过程中的稳定存在。接下来我们将研究目标扩展到整个质粒和宿主,从基因组学的角度研究质粒-宿主的进化历史。2、pMF1质粒和宿主M.fulvus 124B02基因组组学研究暗示了两者的共进化。我们对pMF1质粒上的23个基因所编码的蛋白分别进行功能来源预测,并归为四类。其中14个蛋白与粘细菌密切相关,质粒上约1/3(8个)的编码蛋白只能与Mstipitatus DSM14675比对到同源蛋白,另外,1个来自于Stigmatella aurantiaca,1个来自于Anaeromyxobacter,1个来自于Chondromyces crocatus和Sorangium cellulosum,3个来自于粘细菌众多种属。9个pMF1蛋白在数据库中比对不到任何的同源蛋白,属于pMF1特有。但是,很多蛋白的功能仍然未知。转录组数据表明在23个基因中,转录水平最高的是pMF1.17,pMF1.18,其次是pMF1.12。链特异性转录组和RT-PCR结果表明pMF1包含6个操纵子,占全部基因比例的87%(20/23)。接下来我们对宿主M. fulvus 124B02进行了基因组全测序,结果表明M. fulvus 124B02包含一个环形染色体,大小为11,048,835 bp,以及一个环形质粒,也就是pMF1。染色体和质粒基因组的GC含量相似,分别为69.96%和68.7%。全基因组进化树和共线性比对表明M.fulvus 124B02与M.stipitatus DSM14675同源性最高,二者在基因组大小上也最接近。与其他粘球菌相比,M.fulvus 124B02的基因组有1-2 Mb的扩张,但其在直系同源和旁系同源基因比例上并没有明显差异。限制修饰系统和CRISPR-Cas系统比较分析发现,M.fulvus 124B02的防御系统更加薄弱,其Cas蛋白操纵子比其他粘球菌要少1/2-2/3,记录外源DNA的spacers也少于其他同种或同属的粘细菌,限制修饰系统类型和修饰酶种类也较少。同源比对结果显示pMF1上的某些基因来自于其他粘细菌,暗示了pMF1曾经在不同粘细菌之间水平转移,而且与M.stipitatus DSM14675的同源基因最多。粘球菌属产生于47-51百万年前,而M.fulvus 124B02与M.stipitatus DSM14675在大约41百万年前时由共同祖先分化而来,相对薄弱的免疫系统解释了为什么pMF1最终在M.fulvus 124B02中保存下来,并与M.fulvus 124B02共同进化,稳定存在。3、pMFl在宿主M.fulvus 124B02中发挥维持宿主基因组稳定的作用为了探明pMF1稳定存在于M.fulvus 124B02中的机制,我们构建了质粒消除菌株,在实验室条件下模拟pMF1与宿主M.fulvus 124B02的进化。利用质粒不相容原理可以将pMF1自M.fulvus 124B02中消除,而且pMF1的消除没有显著影响宿主的生长、运动、发育等表型,说明pMF1对宿主的影响并不是短时间的表型影响。在进行实验室传代时,我们设定了三种培养条件,分别以丰富的CYE、贫瘠的dead cells和捕食性的living cells为食物来源。结果发现只有在以贫瘠的dead cells为营养时,pMF1能稳定存在,而在其他两种条件下传代的菌株中,pMF1在7-8周时便检测不到。为了找到影响pMF1稳定存在的相关基因,我们对三种条件传代的菌株进行测序,对筛选出的基因进行敲除,重复传代实验,最终确定了可能相关的一些基因。我们筛选到了pMF1稳定存在的实验室条件,在该条件下,对经过较长时间共进化的菌株进行表型分析时发现不携带质粒的菌株其发育能力下降程度要明显高于携带质粒的菌株,CYE平板上的124B02/free菌株甚至由聚团生长变成分散生长,暗示了pMF1对宿主的影响可能是经过长时间的逐渐积累。为了验证这一猜想,我们对进化菌株进行基因组测序,结果表明124B02/free菌株的基因组突变率要明显高于124B02/4111和124B02/pMF1菌株,pMF1或者pZJY4111质粒的存在能降低基因组突变率,而且这些突变的发生是随机的,没有基因组位置和基因功能的偏好性。pMF1和pZJY4111的共同部分是质粒的ori和par loci,而pZJY4111质粒在进化传代过程中ori被宿主剪切,只剩下par loci的现象表明可能是par loci发挥了稳定基因组,防止基因组发生突变的作用。综上所述,pMF1的发现,不仅成功解决了粘细菌的遗传操作问题,同时为我们了解粘细菌基因组进化提供了指导。据此,我们推测了pMF1隐秘质粒稳定存在的机制模型。在粘细菌的进化历史过程中,做为基因移动的载体,pMF1曾经在粘细菌之间水平转移。由于结构简单、拷贝数高,质粒被认为是快速的基因进化器,可以加速宿主基因组的进化。由于具有相对较弱的免疫防御系统,pMF1更容易进入M.fulvus 124B02宿主细胞内。pMF1通过参与宿主基因组的错配修复过程,最终被M.fulvus 124B02需要而保存下来。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
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本文编号：1884189