Probing DNA segregation in archaea: molecular dissection of an atypical tricistronic partition system from Sulfolobus

探索古细菌中的 DNA 分离：来自硫化叶菌的非典型三顺反子分配系统的分子解剖

• 批准号: BB/F012004/1
• 负责人: Daniela Barillà
• 金额: $ 44.38万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF012004%2F1
• 关键词: Probing; DNA; segregation; archaea; molecular

The process of genome segregation is a fundamental stage of the life cycle of each cell: the genetic content is first duplicated, then separated and equally distributed into the two daughter cells. The mechanism whereby the genetic material (that is organized into linear DNA chromosomes) is separated in cells of higher organisms (like plants, animals, humans) has been extensively studied and is well understood. In these cells the molecular machine responsible for chromosome segregation is known as 'mitotic spindle': it consists of cables, called microtubules, which are anchored to chromosomes at a specific site, known as 'centromere'. The microtubules pull sister chromosomes to opposite poles of the spindle, before the cell divides. In bacteria the picture is more elusive. The genetic patrimony of bacteria consists of a single circular (more rarely linear) chromosome and sometimes smaller circles of DNA called plasmids. Historically, it was presumed that segregation of bacterial chromosomes and plasmids was a passive process, not requiring a dedicated apparatus and perhaps involving attachment of the newly-replicated genetic elements to the growing cell wall. However, in recent years evidence has been provided pointing to the existence of an active mechanism responsible for the segregation of chromosomes and plasmids, requiring the participation of dedicated factors. In bacteria, the most well-characterized DNA segregation systems are those specified by plasmids, which are present in the cell in low numbers. These plasmids harbour their own survival kit, a segregation cassette consisting of two genes, often termed parA and parB, and a centromere-like site. This cassette ensures an accurate segregation of the plasmids from one generation to the next at cell division. The molecular mechanisms underlying this process have not been thoroughly elucidated as yet; however, recent discoveries hint at the existence of mitotic spindle-like machineries. Archaea are the third domain of life: their discovery in 1977 represented a major biological milestone. They were initially identified as a different group of organisms on the basis of sequences contained in their RNA or ribonucleic acid. Further characterization of their physiology, biochemistry and genetics has provided unequivocal evidence that they are a distinct group of organisms (different from bacteria and from higher multicellular organisms). The first archaea to be studied were all from extreme environments, but they are now known to thrive in most of the earth's ecological niches and they constitute ~20% of the biosphere. Hyperthermophilic archaea grow at 80 C and above and exhibit unusual properties, which make these organisms a valuable resource for the development of novel biotechnological processes. Industrial applications include the production of archaea-derived enzymes, which are stable at high temperature, cellulose degrading enzymes, the use of their membranes as delivery systems for drugs and genes. Despite numerous studies on fundamental biological processes in archaea, to date no information is available on the mechanism of genome segregation in these organisms. We intend to analyze this process in an archaeon called Sulfolobus NOB8H2, which has been isolated from hot springs in the island of Hokkaido, Japan. This archaeon contains a plasmid, pNOB8, which harbours a putative DNA segregation cassette comprising three genes (designated as orf44, parB, parA). The project here proposed will focus on the characterization of the factors encoded by the genes above, their function and respective role in pNOB8 partitioning at cell division and their dynamic interactions. We also intend to identify the centromere of pNOB8 and dissect the interactions between this site and ParB, ParA and perhaps Orf44. Furthermore, investigations will be conducted to probe whether the ParA protein assembles into cable-like structures as observed for some bacterial ParA counterparts.

基因组分离的过程是每个细胞生命周期的一个基本阶段：遗传内容首先复制，然后分离并平均分配到两个子细胞中。遗传物质（组织成线性DNA染色体）在高等生物（如植物，动物，人类）细胞中分离的机制已被广泛研究并得到充分理解。在这些细胞中，负责染色体分离的分子机器被称为“有丝分裂纺锤体”：它由称为微管的电缆组成，这些电缆在称为“着丝粒”的特定位点锚定到染色体上。在细胞分裂之前，微管将姐妹染色体拉到纺锤体的两极。在细菌中，情况就更难以捉摸了。细菌的遗传遗产由一个单一的环状（很少是线性的）染色体和有时更小的称为质粒的DNA环组成。从历史上看，人们认为细菌染色体和质粒的分离是一个被动的过程，不需要专门的仪器，可能涉及将新复制的遗传元件附着到生长的细胞壁上。然而，近年来已经提供的证据表明存在一种负责染色体和质粒分离的主动机制，需要专用因子的参与。在细菌中，最具特征的DNA分离系统是那些由质粒指定的系统，质粒以低数量存在于细胞中。这些质粒携带有它们自己的生存试剂盒，一个由两个基因组成的分离盒，通常称为帕拉和parB，以及一个着丝粒样位点。该盒确保在细胞分裂时质粒从一代到下一代的准确分离。这一过程的分子机制尚未完全阐明，然而，最近的发现暗示了有丝分裂纺锤体样机制的存在。第三个领域是生命：它们在1977年的发现代表了一个重要的生物学里程碑。它们最初被确定为一个不同的生物群，其基础是它们的RNA或核糖核酸中所含的序列。对它们的生理学、生物化学和遗传学的进一步表征提供了明确的证据，证明它们是一个独特的生物群体（不同于细菌和高等多细胞生物）。第一批被研究的古生菌都来自极端环境，但现在已知它们在地球上大多数生态位中茁壮成长，占生物圈的20%。超嗜热古菌在80 ℃及以上生长，并表现出不寻常的特性，这使得这些生物体成为开发新型生物技术过程的宝贵资源。工业应用包括生产在高温下稳定的古细菌衍生酶、纤维素降解酶、将其膜用作药物和基因的递送系统。尽管对古生菌的基本生物学过程进行了大量研究，但迄今为止还没有关于这些生物体中基因组分离机制的信息。我们打算分析这一过程中的古菌称为Sulfolobus NOB 8 H2，已被隔离的温泉在日本的北海道。该古细菌含有质粒pNOB 8，其具有包含三个基因（命名为orf 44、parB、帕拉）的推定DNA分离盒。本项目将重点研究上述基因编码的因子的特性、它们在细胞分裂时pNOB 8分配中的功能和各自的作用以及它们之间的动态相互作用。我们还打算鉴定pNOB 8的着丝粒，并分析该位点与ParB、帕拉和可能的Orf 44之间的相互作用。此外，将进行调查，以探测是否帕拉蛋白组装成电缆样结构，观察到一些细菌帕拉对应物。

项目成果

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.

古细菌 DNA 分离机制的结构揭示了细菌和真核生物的联系。

• DOI: 10.1126/science.aaa9046
• 发表时间: 2015-09-04
• 期刊: Science (New York, N.Y.)
• 作者: Schumacher MA;Tonthat NK;Lee J;Rodriguez-Castañeda FA;Chinnam NB;Kalliomaa-Sanford AK;Ng IW;Barge MT;Shaw PL;Barillà D
• 通讯作者: Barillà D

Driving Apart and Segregating Genomes in Archaea.

• DOI: 10.1016/j.tim.2016.07.001
• 发表时间: 2016-12
• 期刊: TRENDS IN MICROBIOLOGY
• 影响因子: 15.9
• 作者: Barilla, Daniela

Bacterial Chromatin

细菌染色质

• DOI: 10.1007/978-90-481-3473-1_4
• 发表时间: 2010
• 作者: Hayes F

One-way ticket to the cell pole: plasmid transport by the prokaryotic tubulin homolog TubZ.

通往细胞极的单程票：通过原核微管蛋白同系物 TubZ 进行质粒运输。

• DOI: 10.1073/pnas.1007331107
• 发表时间: 2010
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Barillà D