Genetics and regulation of surface structures in the Domain Archaea

古细菌是原核生物，与细菌和真核生物不同，被归类为生命的三个领域之一。他们的发现被誉为上个世纪最激动人心的科学发现之一。古生菌最初因其在代表地球上生命极限的极端栖息地中茁壮成长的能力而臭名昭著，但现在已知它们在海水、土壤和人体中无处不在且数量丰富。古生菌现在被认为在自然界的碳、氮和硫循环中扮演着重要的角色。对于许多古生物来说，与其自然环境的相互作用是通过各种不寻常的表面附属物，包括古生菌(以前称为古生鞭毛)和菌毛，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-连接的多糖而被修改，这是在同等的细菌结构中没有观察到的一种新的古生菌特有的特征，以及一种被认为有助于在古生菌经常繁盛的极端环境中保持蛋白质稳定性的修饰。我们是第一个对古生菌和N-连接糖基化进行遗传学研究的实验室，鉴定了许多参与这种独特的糖的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。我们已经确定了含有一种在自然界其他地方没有报道的糖的新型多糖结构，以及在不断增长的定制糖工程领域中可能有重要应用的基因。目前的应用旨在扩大我们对N-连接的糖基化及其在表面附属物组装和功能中的作用的研究，通过鉴定参与单个糖链糖组分的生物合成的其他基因以及它们组装成最终的聚糖体，同时还定义附属物形成和功能所需的糖基化位点的位置和最小数目。我们还将利用我们的模式生物--马里帕鲁斯甲烷球菌，开始研究控制附件合成的环境条件。我们提出了一项计划，通过比较不同突变株的完整基因组序列来识别控制这些重要表面结构合成的调节子，这些突变株由于不能转录相关基因而无法组装古菌。这些研究将使用在我的实验室中已经成熟的遗传学、电子显微镜和生化方法，以及旨在提高将进行拟议实验的研究生的技能的新技术。这些研究将有助于完善我们对maripaludis N-连接糖基化途径的知识，通过我们的努力，Maripaludis已经是古生代研究得最好的途径之一，并填补了我们在表面结构组装如何受到环境调控和影响的知识空白。这些数据将补充其他模式古生菌的补充研究，并有助于阐明为什么糖基化是古菌形成的必要修饰的全球图景。对古生代的研究已经帮助澄清了其他结构域共享的N-糖基化系统的某些方面。参与这些项目的研究生将学习各种分子生物学和生化技术，以及处理一些已知的严格微生物厌氧菌的培训，并有机会通过访问我的合作者的实验室获得其他现代实验室技能，然后将这些技能转移到我的研究团队。这些技能可转移到基础、应用、生物技术和医疗领域的职业。

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.