Genetics and regulation of surface structures in the Domain Archaea

古细菌是原核生物，被归类为与细菌和真核生物不同的三个生命领域之一。他们发现了第三条进化谱系，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，但现在人们知道它们在海水、土壤和人体中无处不在，而且数量丰富。古细菌现在被认为在自然界的碳、氮和硫循环中起着至关重要的作用。对于许多古菌来说，与自然环境的相互作用是通过各种不同寻常的表面附属物，包括古菌（以前称为古菌鞭毛）和毛，这些附属物参与了多种功能，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上连接n链聚糖进行修饰，这是一种新的古细菌特异性特征，在相同的细菌结构中没有观察到，并且已经提出了一种修饰，有助于古细菌在极端环境中茁壮成长的蛋白质稳定性。我们是第一个对古菌和n -链糖基化进行遗传学研究的实验室，鉴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着物组装和功能的重要性。我们已经确定了新的聚糖结构，其中含有自然界中其他地方未报道的糖和基因，这些结构可能在定制糖工程领域有重要的应用。目前的应用旨在通过识别参与单个聚糖糖组分的生物合成及其组装成最终聚糖的其他基因，同时确定附属物形成和功能所需的糖基化位点的位置和最小数量，扩大我们对n -链糖基化及其在表面附属物组装和功能中的作用的研究。我们还将开始研究控制附件合成的环境条件，使用我们的模式生物，马里帕卢迪甲烷球菌。我们提出了一个计划，通过比较各种突变菌株的全基因组序列来确定控制这些重要表面结构合成的调节因子，这些突变菌株由于无法转录相关基因而无法组装古菌。这些研究将采用我的实验室已经建立的遗传学、电子显微镜和生化方法，以及旨在提高将要进行拟议实验的研究生技能的新技术。这些研究将有助于完善我们对M. maripaludis中n -链糖基化途径的了解，这已经是我们在古生菌中研究得最好的途径之一，并填补我们对表面结构组装如何受到环境调节和影响的知识空白。这些数据将增强对其他古菌模型的补充研究，并有助于阐明为什么糖基化是古菌形成的必要修饰。对古细菌的研究已经帮助阐明了其他结构域共有的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化技术，以及处理已知的一些严格的微生物厌氧菌的培训，并有机会通过参观我的合作者的实验室来获得其他现代实验室技能，然后这些技能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中。

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.