Genetics and regulation of surface structures in the Domain Archaea

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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.