Genetics and regulation of surface structures in the Domain Archaea

古细菌领域表面结构的遗传学和调控

• 批准号: RGPIN-2014-06124
• 负责人: Jarrell, Ken
• 金额: $ 3.86万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567610
• 关键词: Genetics; regulation; surface; structures; Domain

Archaea are prokaryotes that are categorized as one of the three Domains of life distinct from Bacteria and Eukaryotes. Their discovery as a third line of evolutionary descent has been hailed as one of the most exciting scientific discoveries of the last century. Archaea first gained notoriety for their ability to thrive in extreme habitats representing the limits for life on Earth but they are now known to be ubiquitous and abundant in ocean waters, soils and in the human body. Archaea are now recognized to play essential roles in the carbon, nitrogen and sulfur cycles in nature. For many Archaea, interaction with their natural environment is through a variety of unusual surface appendages including archaella (formerly, archaeal flagella) and pili, which are involved in a diverse set of functions including attachment, motility, biofilm formation and DNA exchange. These structures are comprised of subunits which have been modified by the attachment of N-linked glycans at several sites, a novel archaeal-specific feature that is not observed in the equivalent bacterial structures and a modification that has been proposed to contribute to protein stability in the environmental extremes that archaea often thrive. We were the first laboratory to genetically investigate archaella and N-linked glycosylation, identifying many genes involved in the assembly and biosynthesis of the unique glycan and demonstrating the importance of this modification for surface appendage assembly and function. We have identified novel glycan structures containing a sugar not reported elsewhere in nature and genes that could have important application in the growing field of tailored glyco-engineering. The current applications aims to extent our studies on N-linked glycosylation and its role in surface appendage assembly and function by identifying additional genes involved in the biosynthesis of the individual glycan sugar components and also their assembly into the final glycan while also defining the location and minimum number of glycosylation sites necessary for appendage formation and function. We will also initiate studies on environmental conditions that control appendage synthesis using our model organism, Methanococcus maripaludis. We propose a plan to identify regulators that control synthesis of these important surface structures, by comparing the complete genome sequences of various mutant strains that are unable to assemble archaella since they cannot transcribe the genes involved. These studies will employ genetic, electron microscopic and biochemical methodologies already well-established in my lab as well as new techniques designed to enhance the skill set of the graduate students who will do the proposed experiments. These studies will help complete our knowledge of the N-linked glycosylation pathway in M. maripaludis, already one of the best studied in Archaea through our efforts, as well as fill in gaps in our knowledge of how assembly of surface structures is regulated and influenced by the environment. These data will augment complementary studies in other model Archaea and help to elucidate a global picture of why glycosylation is a necessary modification for archaella formation. Studies in Archaea have already helped to clarify aspects of N-glycosylation systems shared by the other Domains. Graduate students involved in these projects will learn a variety of molecular biology and biochemical techniques as well as training to handle some of the stringent microbial anaerobes known, with an opportunity to obtain other modern laboratory skills through visits to the labs of my collaborators which can then be transferred to my research team. These skills are transferable to careers in basic, applied, biotechnological and medical fields.

微生物是原核生物，被分类为不同于细菌和真核生物的三个生命领域之一。他们作为第三条进化血统的发现被誉为上个世纪最令人兴奋的科学发现之一。海蛞蝓最初因其在代表地球生命极限的极端栖息地中茁壮成长的能力而声名狼借，但现在已知它们在海洋沃茨、土壤和人体中无处不在。现在，人们认识到，藻类在自然界的碳、氮和硫循环中发挥着重要作用。对于许多古细菌来说，与自然环境的相互作用是通过各种不寻常的表面附属物，包括古菌（以前的古生鞭毛）和皮利，它们参与了一系列不同的功能，包括附着，运动，生物膜形成和DNA交换。这些结构由亚基组成，这些亚基通过在几个位点连接N-连接聚糖进行了修饰，这是一种在等效细菌结构中未观察到的新的古细菌特异性特征，并且已经提出了一种修饰，该修饰有助于在古细菌经常茁壮成长的极端环境中保持蛋白质稳定性。我们是第一个对古菌和N-连接糖基化进行遗传研究的实验室，确定了许多参与独特聚糖组装和生物合成的基因，并证明了这种修饰对表面附属物组装和功能的重要性。我们已经确定了新的聚糖结构，其中含有一种在自然界其他地方没有报道的糖，以及在不断增长的定制糖工程领域可能具有重要应用的基因。目前的申请旨在通过鉴定参与单个聚糖糖组分的生物合成以及它们组装成最终聚糖的其他基因，同时还定义附件形成和功能所需的糖基化位点的位置和最小数量，来扩展我们对N-连接糖基化及其在表面附件组装和功能中的作用的研究。我们还将使用我们的模式生物海沼甲烷球菌启动对控制附属物合成的环境条件的研究。我们提出了一个计划，以确定监管机构，控制这些重要的表面结构的合成，通过比较完整的基因组序列的各种突变株，无法组装古细菌，因为他们不能转录所涉及的基因。这些研究将采用遗传学，电子显微镜和生物化学方法已经在我的实验室以及新技术，旨在提高研究生谁将做拟议的实验的技能。这些研究将有助于完善我们对M中N-连接糖基化途径的认识。maripaludis，已经是通过我们的努力在西海岸最好的研究之一，以及填补了我们的知识空白，表面结构的组装是如何调节和环境的影响。这些数据将增强其他模式古细菌的互补研究，并有助于阐明为什么糖基化是古细菌形成的必要修饰的全球图景。对Escherichia的研究已经帮助澄清了其他结构域共享的N-糖基化系统的各个方面。参与这些项目的研究生将学习各种分子生物学和生物化学技术，以及处理一些已知的严格微生物厌氧菌的培训，有机会通过参观我的合作者的实验室获得其他现代实验室技能，然后可以转移到我的研究团队。这些技能可转移到基础，应用，生物技术和医学领域的职业生涯。