Electron Cryo-microscopy of Archaeal Surface Structures

Abstract

The domain of life known as Archaea, encompasses an astonishing array of intricately diverse species thriving across a wide spectrum of highly varied environments around the globe. First discovered in the 1970s (recognized as archaeabacteria at the time), the uncovering of a collection of methanogenic species initiated the development of the kingdom now known as archaea. From this initial identification of anaerobic species found in thermal springs 1,2 , the search for other extremophile species then began. The subsequent discovery of halophilic species from salt lakes and salt-saturated soils 3 , as well as thermophilic species within hot acidic sulphur pools 4 , led to an array of novel environmentally distinct organisms. Comparisons of these habitually diverse species found both biochemical similarities as well as significant homology between their 16S rRNA and tRNA molecules 5,6 . This ultimately led to the discovery of a defined branch of organisms, distinct from bacteria, that would later be classified as the archaeal kingdom. Whilst archaeal species have conquered some of the most extreme ecological niches on planet earth 7–10 , many archaea have been located within a plethora of ambient environments also 11–14 , contributing to nutrient cycles and performing organic decompositions within mesophilic life systems. This vast success across the archaeal kingdom is due to a list of intricate developments. Genetic flexibility has allowed for key adaptations, promoting success over a vast array of niches. The generation of specialised enzymes, optimally functional across temperatures outside the range of prokaryotic and eukaryotic counterparts, has allowed for growth in many areas unchallenged by other life forms. The utilisation of huge versatile lists of metabolites has also seen archaea populate areas of the globe that no other kingdom of life can. However, one other such key area has also played a pinnacle role, the archaeal surface structures. Within the course of evolution, prokaryotic and thus by association archaeal organisms have developed a broad spectrum of cell enveloping surface structures. The most ubiquitous of these are surface layers (S-layers), which form a proteinaceous lattice completely covering the cell. S-layers are present in almost all archaea, and shared amongst various species of almost every taxonomic group of walled bacteria. These paracrystalline structures provide organisms with selection advantages through provision of a variety of novel functions: acting as protective coats to survive harsh conditions, molecular sieves to select for macromolecule access into the cytoplasm, environments for ion traps to localise metals for functionality, virulence protection, cell shape and division control measures 15–18 . In association with the S-layer, archaea also produce a variety of filamentous appendages that protrude through it. Providing a number of distinct biological functions such as adhesion, biofilm formation, motility, species-species interaction, as well as the exchange of genetic material between cells 19,20 . The aims within this PhD thesis were firstly to obtain a deeper understanding of the arsenal of filamentous appendages within a single specific archaeal model organism, Sulfolobus acidocaldarius. This was achieved by employing Electron Cryogenic Microscopy (CryoEM), which then revealed the structures of three distinct filaments – archaella, archaeal-adhesive pili (Aap) and threads. Leveraging bioinformatics, these three structures and by association, their machinery complexes were then analysed in correlation with each other and in relation to the archaeal S-layer. Thereby offering valuable insights into their evolutionary implications. Fundamentally, this data now provides insight in to how these structures link to other species, primarily within the archaeal world, but additionally how they relate to bacterial filaments. In addition, two evolutionary distinct and S-layer-less species of the phylum Thermoplasmatales, Cunicuplasma divulgatum and Oxyplasma meridanum were investigated. The subsequent structural findings shed new light on the question whether archaea lacking S-layers produce canonical or divergent archaella and pili. An emphasis was placed on the filament operons of these species, providing both genetic and structural insights into how archaeal filament machinery differs between filament types, as well as between species of Archaea from different Phyla. The overarching objective of this thesis was to contribute a piece to our ongoing efforts in unravelling the mysteries of archaeal life.