Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses - which infect members of the order Sulfolobales, growing optimally at 80º C and pH 3 - are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. They provide a malleable and useful genetic tool for probing into the functions of their host, as well as the host responses to infection. Nonetheless, much about these viruses remains to be learned to further understand their morphological, genetic, and life cycle characteristics. In order to investigate these aspects of these Fuselloviridae, as well as their evolution, this work reports the isolation and characterization of a novel fusellovirus, Sulfolobus Spindle-shaped virus 10 (formerly SSV-L1). Genetic and genomic analyses highlight significant homology with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF which may play a role in anti-CRISPR host evasion. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1. Lastly, analysis of predicted protein structures from SSV10 provide new insights into virus-host interactions. These analyses help to expand our understanding of the viral life cycle while contextualizing the mutagenesis data presented in the following chapters.

Thesis (Ph.D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: C. Martin Lawrence. Includes bibliographical references (leaves 146-159).

Thesis (M.S.)--University of North Carolina at Wilmington, 2003.

Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Mensur Dlakic. Includes bibliographical references (leaves 56-61).

Thesis (MS)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: Brian Bothner. Includes bibliographical references (leaves 63-71).

Thesis (Ph.D.) - University of St Andrews, March 2008.

All sequenced archaeal genomes encode a general transcription factor, TFE, which is highly conserved and homologous to the alpha subunit of the eukaryotic transcription factor TFIIE. TFE functions to increase promoter opening efficiency during transcription initiation, although the mechanism for this is unclear. The N-terminus of TFE contains a common DNA binding motif, a winged helix. At the tip of this winged helix is a highly conserved region of aromatic amino acids that is close to DNA during initiation. TFE activation can compensate for mutations in another transcription factor, TFB2, which is homologous to TFIIB. P. furiosus encodes two paralogs of the eukaryotic RNA polymerase II transcription factor TFIIB: TFB1 and TFB2. TFB2 lacks a portion of the highly conserved N-terminus, and functions in transcription complexes at a lower efficiency than TFB1. It has been demonstrated that the presence of TFE is able to assist in transcription with TFB2 in vitro bringing its efficiency to almost TFB1 levels. Thus, TFB2 provides a unique opportunity to evaluate the function of the TFE winged helix in transcription. In this study the aromatic patch of the TFE winged helix was mutated to test its role in activation of TFB1 and TFB2-containing transcription complexes, because this aromatic patch is required for full TFE activity especially when NTP concentrations are low.

The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is unusual as it is missing highly conserved sequences in its N-terminal domain that are present in TFIIB and TFB1. Despite this, TFB2 is effective in transcription process, albeit with lower efficiency compared to TFB1. Other archaea also contain multiple TFBs, but unlike Pyrococcus furiosus TFB2, these multiple TFBs have higher sequence homology to each other and have similar transcription efficiencies. Photochemical cross-linking experiments have shown that the B-reader of TFB in archaea and TFIIB in eukaryotes is close to transcription start site and is very important in RNAP recruitment to promoter DNA and transcription start site selection. Thus the lack of the highly conserved B reader region in P. furiosus TFB2 presents the opportunity to further study the functional importance of this region. In this study several amino acids in N-terminal domain of TFB2 were mutated with photoactivable unnatural amino acid p-benzoyl L- phenylalanine (pBpa) and the proximity of TFB2 relative to DNA was determined by photochemical cross-linking experiments. The results showed that TFB2 interacts with DNA near the TATA box via its C-terminal domain, and interacts with both strands of DNA near the transcription start site via its divergent B-reader and the B-linker sequences. The B-reader loop region is close to transcription start site and interacts with the transcribed strand of promoter DNA while the B-linker strand cross-links with the non-transcribed strand. Some of the amino acids in between the B-reader loop and the B-linker strand region in TFB2 are seen to cross-link both the transcribed and the non-transcribed strand. Thus, despite the absence of strong homology to conserved B-reader and B-linker sequences, TFB2 is likely to interact with DNA in the transcription bubble and facilitate in transcription initiation.

The biosynthesis of caldariellaquinone (CQ) has been studied in Sulfolobus acidocaldarius using a variety of methods. By growing cells with a series of tyrosines labeled with deuterium or ¹³C and measuring the extent and position at which label was incorporated into the CQ by mass spectrometry, it was concluded that the benzo[b]thiophen-4,7-quinone ring of CQ is derived as an intact unit from all of the carbons of tyrosine except C-1. Additional work, using (3S)-L-(2-²H, 3-²H]-, (3R)-D-[2-²H, 3-²H]-, (3S)-D-[3-²H]-, and (3R)-L-[3-²H]- tyrosine, demonstrated that the pro-3S hydrogen of either D- or L-tyrosine is the origin of the C-3 proton of the benzo[b]thiophene ring. Considering the above information and the structure of CQ, it was concluded that CQ was most likely biosynthesized by the condensation of farnesylfarnesyl pyrophosphate with homogentisic acid (HA) in a reaction analogous to that found in the biosynthesis of ubiquinone. The possibility of this reaction being involved in the biosynthesis of CQ was supported by the identification of farnesylfarnesol, a hydrolytic breakdown product of farnesylfarnesyl pyrophosphate, by gas chromatography-mass spectrometry (GC-MS) of purified lipid extracts. The possible involvement of HA in CQ biosynthesis, however, could not be confirmed by five independent methods. The possible formation of CQ by the condensation of benzo[b]thiophen-4,7-quinone with farnesylfarnesyl pyrophosphate was eliminated by the inability to detect benzo[b]thiophen-4,7-quinone in S. acidocaldarius. Attempts to identify the tyrosine metabolites leading to CQ by studing the metabolisms of tyrosine, 2-fluorotyrosine, and 3-fluorotyrosine in S. acidocaldarius lead to the identification of two previously undescribed pathways for tyrosine metabolism. These two pathways branch after the conversion of tyrosine to 4-hydroxyphenylacetic acid (pHPA). The ability of labeled pDHPA to be incorporated into these metabolites, but not into CQ, indicates that the first committed step in the biosynthesis of CQ occurs at either tyrosine or a metabolite very closely related to tyrosine, e.g., 4-hydroxyphenylpyruvate (pHPP). Analysis of the extract of the cells grown with 3-fluorotyrosine showed two fluorine-containing compounds, which are likely to be fluoro-analogues of the intermediates in the biosynthesis of CQ. However, because of the small amount of these two compounds found (24 nmoles/g of wet weight), structural characterization was not possible. Both the methyl and sulfur groups of the methylthio portion of CQ were shown to arise from methionine. Mass spectral analysis of the CQ isolated from cells grown in the presence of [³⁴S-methyl-²H₃]-L-methionine clearly showed, however, that the methylthio group of CQ is not derived as an intact unit from the methylthio group of methionine. Additional work supported the theory that the methionine sulfur first undergoes transsulfuration to cysteine, which then supplies the sulfur for both the methylthio and the benzo[b]thiophene moieties of CQ. This represents the first example of transsulfuration from methionine to cysteine occurring in archaebacteria. / Ph. D.

The need to develop a renewable, environmentally friendly source of energy has become a primary focus in modern science, with bio gas showing considerable potential. Interest in the methanogenic Archaea has therefore grown in recent years and extensive studies have been carried out to investigate the population diversity in various habitats. Presently, there are only a few studies that have evaluated the microbial communities inhabiting the gastrointestinal tract of wildlife native to southern Africa. This study aimed to investigate the microbial diversity, in particular the bacterial and methanogen communities involved in fermentative digestion in the gastrointestinal tract of zebra. Assessment of the microbial diversity in zebra faeces included both culture-based techniques and nucleic acid targeting analysis via 16S rRNA gene sequencing. Quantitative analysis using selected solid media revealed high counts for aerobic and anaerobic Bacteria (7.51x108 and 2.45x109/gram of faecal sample respectively). The majority of aerobic colonies that were detected exhibited Bacillus-like morphology. Nucleic acid based analysis of the diversity of both Bacteria and methanogenic Archaea in zebra faecal material was performed. Both manual and kit based extractions were used for DNA isolation in order to compare the efficiency of the two methods. Results show that a vigorous mechanical treatment was best for the release of DNA from the faecal matter. Amplification of target gene regions was carried out using established primer pairs (ARCH69F/ARCH915R and EUB338F/EUB907R) for methanogen and bacterial DNA respectively. Amplified 16S rRNA gene regions were cloned into a high copy number vector and random clones were selected for evaluation. Clones containing the target gene were further analysed by ARDRA and were assigned to a specific phylotype. Two bacterial (105 clones in total) and three methanogen (178 clones in total) clone libraries were constructed, of which 24 phylotypes were established for Bacteria and 25 for methanogenic Archaea. A representative of each phylotype was analysed by sequencing and further phylogenetic analysis was conducted. Six bacterial phylotypes, which represented 56% of all bacterial clones, exhibited 99% sequence similarity to Bacillus species. Six methanogen phylotypes, which exhibited 99% sequence similarity to the hydrogenotrophic species Methanobrevibacter gottschalkii strain PG, were established to be predominant in zebra faeces. These phylotypes represented 71% of all archaeal clones selected for analysis in this study. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.