A novel DNA segregation model system from Archaea revealing bacterial and eukaryotic linkages

古生菌是在数十亿年前作为生命的一个领域进化而来的，但它们是在生物的普遍树的地图上相对较新的成员。它们在40年前的发现是一个重要的里程碑。古细菌是与细菌和真核生物一起生活在地球上的单细胞生物。细菌和古细菌都是原核生物，也就是说，它们的遗传物质并没有被膜包裹在一个叫做细胞核的单独的隔间里，而细胞核是真核生物（如面包酵母、真菌、植物、动物和人类）的标志。众所周知，古细菌无处不在，构成了生物圈的相当一部分。它们的普遍存在和丰富程度使它们成为调节地球生物地球化学循环的关键角色。从功能和机制的角度来看，古细菌是细菌和真核生物特征的马赛克，但它们也具有独特的特征，如甲烷生产。喜热古细菌是一种超级微生物，在80摄氏度或更高的温度下，在温泉、火山、深海喷口中繁衍生息，并表现出不同寻常的特性，这使得这些生物对开发新的生物技术应用有价值，但对极端生命的研究也非常有趣。它们在其他陆地生物无法生存的极端环境中生长的能力，也重新燃起了发现外星生命的希望。尽管在过去的四十年里，在解码这些生物的分子机制方面取得了重大进展，但迄今为止，关于古细菌DNA分离的基本过程的信息很少，这个主题仍然是一个等待调查的黑匣子。基因组分离是每个细胞生命周期的关键阶段：DNA首先被复制，然后被分离，并平均分配到两个子细胞中。我们打算在一株从日本酸性温泉中分离出来的喜热古菌Sulfolobus中研究这一过程。这种微生物含有一个大的和一个小的DNA环。大的称为染色体，小的称为质粒。我们最近研究了三种由质粒pNOB8编码的蛋白质，并解决了它们的三维结构。这些蛋白质组装成一个纳米机器，驱动复制的姐妹质粒分开，这样每个子细胞得到相同的拷贝数。我们打算在宿主Sulfolobus菌株的DNA中引入变化，以便在编码蛋白质的基因中引入突变，以表明这些因素对质粒的遗传是必不可少的。负责pNOB8遗传的蛋白质之一是AspA。它与质粒上的一个特殊位点紧密结合，作为对接位点，然后与DNA上扩散的邻近区域结合，形成螺旋结构。我们想研究质粒是否包含AspA的多个对接位点，以及AspA蛋白在DNA上延伸的过程。质粒分离纳米机器的另外两种蛋白质是ParB和ParA。ParB是位于复合物中AspA和ParA之间的适配器。接头需要柔韧，而ParB确实是柔韧的：这种蛋白质由两个由柔韧接头连接的结构域组成。其中一个ParB结构域具有细菌的味道，而另一个看起来像真核蛋白。我们怀疑结合ParA的ParB区域是柔性连接体，我们想验证这一假设。我们打算研究Sulfolobus中的其他蛋白是否与ParB的真核结构域结合，并对其进行修饰和调节。最后，我们渴望获得Sulfolobus细胞中这些蛋白质的快照。我们将使用显微镜来提供蛋白质复合体的高分辨率图像，并告诉我们它相对于染色体DNA质量的位置。这种分析将允许识别模式，这将阐明pNOB8 DNA分离纳米机器的寿命。

Archaea evolved as a domain of life billions of years ago, but they are a relatively recent addition to the map of the universal tree of living organisms. Their discovery 40 years ago represented a major milestone. Archaea are unicellular organisms that populate our planet together with bacteria and eukaryotes. Both bacteria and archaea are prokaryotes, i.e. their genetic material is not wrapped by a membrane into a separate compartment, called nucleus, which is instead a hallmark of eukaryotes (baker yeast, fungi, plants, animals and humans to mention some). Archaea are known to be ubiquitous, constituting a considerable fraction of the biosphere. Their ubiquity and abundance make them key players in regulating biogeochemical cycles on Earth. From a functional and mechanistic standpoint, archaea are a mosaic of features from bacteria and eukaryotes, but they are also characterized by unique features like methane production.Heat-loving archaea are super microbes thriving at 80 degrees C and higher temperatures in hot springs, volcanoes, deep sea vents and exhibiting unusual properties, which make these organisms valuable for the development of novel biotechnological applications, but also extremely interesting for studies on life pushed to extremes. Their ability to grow in extreme environments where no other terrestrial organism can survive has also rejuvenated hopes of discovering extraterrestrial life. Despite the significant progress made in decoding molecular mechanisms in these organisms in the last four decades, to date little information is available on the fundamental process of DNA segregation in archaea and the subject remains a black box awaiting investigation. Genome segregation is a crucial stage of the life cycle of every cell: the DNA is first duplicated, then separated and equally distributed into the two daughter cells. We intend to study this process in a strain of the heat-loving archaeon Sulfolobus isolated from an acidic hot spring in Japan. This microbe contains a large and a small ring of DNA. The large is called chromosome and the small is designated as plasmid. We have recently investigated three proteins that are encoded by the plasmid, pNOB8, and solved their three-dimensional structures. These proteins assemble into a nanomachine that drives duplicated sister plasmids apart, so that each daughter cell receives the same copy number. We intend to introduce changes in the DNA of the host Sulfolobus strain, so that mutations can be introduced in the genes encoding the proteins to show that these factors are essential for the inheritance of the plasmid. One of the proteins responsible for the inheritance of pNOB8 is AspA. It binds strongly to a special site on the plasmid that acts as a docking site and then associates to adjacent regions spreading on the DNA and forming a helix. We want to investigate whether the plamid contains multiple docking sites for AspA and the process through which this protein stretches on the DNA. The other two proteins of the plasmid segregation nanomachine are ParB and ParA. ParB is an adaptor sitting between AspA and ParA in the complex. Adaptors need to be pliable and ParB is indeed flexible: the protein consists of two domains connected by a flexible linker. One of the ParB domains has a bacterial flavour, whereas the other looks like a eukaryotic protein. We suspect that the ParB region that binds ParA is the flexible linker and we want to test this hypothesis. We intend to investigate if other proteins in Sulfolobus associate with the eukaryotic domain of ParB, modifying and regulating it. Finally, we are eager to gain a snapshot of these proteins in Sulfolobus cells. We are going to use microscopes that will provide high-resolution images of the protein complex and will tell us where it localizes relative to the DNA mass of the chromosome. This analysis will allow to identify patterns that will shed light on the life of the pNOB8 DNA segregation nanomachine.