Several proteins play a crucial role in the formation of the pre-replication complex (pre-RC) in eukaryotes: origin recognition complex (ORC), CDC6 and minichromosome maintenance proteins (MCM). In yeast, these proteins associate with DNA in a sequence-specific manner during the cell cycle. Archaea, the third kingdom of life, are believed to replicate DNA in a eukaryote-like fashion. Most archaeal species encode single MCM and two CDC6/ORC proteins. The reduced number of replication proteins suggests that archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication. This thesis describes a biochemical analysis of the proteins involved in the formation of pre-replication complex (pre-RC) in Archaea. The yeast two-hybrid system was employed to identify protein-protein interactions within the pre-replication complex of two archaeal species Archaeoglobus fulgidus and Aeropyrum pernix. A library of truncated MCM proteins was used to identify a region that appears to be responsible for hexamerisation of MCM. The pre-RC proteins of archaea belong to the AAA+ family of ATPases. AAA+ proteins function as ATP-driven conformational switches that can mediate the assembly, remodelling, or disassembly of protein complexes. Thus, the assembly of the replication initiation complex may involve an ordered sequence of ATP coupled conformational changes. A variety of biochemical techniques were used to investigate the relationship between ATP hydrolysis, DNA binding and protein interactions within the pre-RC of Archaea.

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Archaea represent one of the three domains of life and were once thought to be restricted to extreme environments. This view has been dramatically changed over the last two decades through the discovery of abundant novel lineages of non-extremophilic archaea, their implication in the global nitrogen cycle and their laboratory cultivation. The recently-described phylum Thaumarchaeota includes autotrophic ammonia oxidisers, thus performing the rate-limiting step of nitrification, and frequently outnumbering their bacterial counterparts particularly in acidic soils. Nitrification is a fundamental component of the global nitrogen cycle and leads to significant fertiliser loss and atmospheric and groundwater pollution. Nitrification rates in acidic soils (pH<5.5), which comprise 30% of the world's soils, equal or exceed those of neutral soils. Paradoxically, autotrophic ammonia oxidizing bacteria and archaea, which perform the first stage in nitrification, do no grow in suspended liquid culture below pH 6.5, where ammonia availability is reduced by ionisation. This thesis describes the identification of pH as a major driver of Group 1.1 c thaumarchaeal abundance and diversity. Furthermore, it reports the cultivation of the first obligately acidophilic ammonia oxidiser, Candidatus Nitrosotalea devanaterra. This organism grows in the pH range 4.0 - 5.5 and represents a previously uncultivated lineage distributed across the globe in acidic soils. The physiology of Candida/us N. devanaterra was characterised, including the influence of organic acids (glyoxylate, pyruvate, oxaloacetate and a-ketoglutarate) on the growth.

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