Characterisation of proteins involved in CRISPR-mediated antiviral defence in Sulfolobus solfataricus

One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis

The structure of Sulfolobus solfataricus 2-keto-3-deoxygluconate kinase

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and utilizes an unusual promiscuous nonphosphorylative Entner-Doudoroff pathway to metabolize both glucose and galactose. It has been proposed that a part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus, in which the 2-keto-3-deoxygluconate kinase (KDGK) is promiscuous for both glucose and galactose metabolism. Recombinant S. solfataricus KDGK protein was expressed in Escherichia coli, purified and crystallized in 0.1 M sodium acetate pH 4.1 and 1.4 M NaCl. The crystal structure of apo S. solfataricus KDGK was solved by molecular replacement to a resolution of 2.0 A and a ternary complex with 2-keto-3-deoxygluconate (KDGlu) and an ATP analogue was resolved at 2.1 angstrom. The complex suggests that the structural basis for the enzyme's ability to phosphorylate KDGlu and 2-keto-3-deoxygalactonate (KDGal) is derived from a subtle repositioning of residues that are conserved in homologous nonpromiscuous kinases.</p

A hydrophobic ammonia-oxidizing archaeon of the Nitrosocosmicus clade isolated from coal tar-contaminated sediment

A wide diversity of ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota exists and plays a key role in the N cycle in a variety of habitats. In this study, we isolated and characterized an ammonia-oxidizing archaeon, strain MY3, from a coal tar-contaminated sediment. Phylogenetically, strain MY3 falls in clade 'Nitrosocosmicus' of the thaumarchaeotal group I.1b. The cells of strain MY3 are large 'walnut-like' cocci, divide by binary fission along a central cingulum, and form aggregates. Strain MY3 is mesophilic and neutrophilic. An assay of (13) C-bicarbonate incorporation into archaeal membrane lipids indicated that strain MY3 is capable of autotrophy. In contrast to some other AOA, TCA cycle intermediates, i.e. pruvate, oxaloacetate and α-ketoglutarate, did not affect the growth rates and yields of strain MY3. The attachment of cells of strain MY3 to XAD-7 hydrophobic beads and to the adsorbent vermiculite demonstrated the potential of strain MY3 to form biofilms. The cell surface was confirmed to be hydrophobic by the extraction of strain MY3 from an aqueous medium with p-xylene. Our finding of a strong potential for surface attachment by strain MY3 may reflect an adaptation to the selective pressures in hydrophobic terrestrial environments

A hydrophobic ammonia-oxidizing archaeon of the Nitrosocosmicus clade isolated from coal tar-contaminated sediment

A wide diversity of ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota exists and plays a key role in the N cycle in a variety of habitats. In this study, we isolated and characterized an ammonia-oxidizing archaeon, strain MY3, from a coal tar-contaminated sediment. Phylogenetically, strain MY3 falls in clade 'Nitrosocosmicus' of the thaumarchaeotal group I.1b. The cells of strain MY3 are large 'walnut-like' cocci, divide by binary fission along a central cingulum, and form aggregates. Strain MY3 is mesophilic and neutrophilic. An assay of (13) C-bicarbonate incorporation into archaeal membrane lipids indicated that strain MY3 is capable of autotrophy. In contrast to some other AOA, TCA cycle intermediates, i.e. pruvate, oxaloacetate and α-ketoglutarate, did not affect the growth rates and yields of strain MY3. The attachment of cells of strain MY3 to XAD-7 hydrophobic beads and to the adsorbent vermiculite demonstrated the potential of strain MY3 to form biofilms. The cell surface was confirmed to be hydrophobic by the extraction of strain MY3 from an aqueous medium with p-xylene. Our finding of a strong potential for surface attachment by strain MY3 may reflect an adaptation to the selective pressures in hydrophobic terrestrial environments

The Scottish Structural Proteomics Facility:targets, methods and outputs

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology