Based on what we have found, we proposed a model for Ni presenting by HypB involved in hydrogenase maturation. HypB binds two Ni ions in the apo- and GDP-bound form. Ni binding also induces dimerization of HypB. Upon GTP binding, HypB can bind an extra Ni ion at the dimeric interface. GTP hydrolysis will release the extra Ni ion, which may be subsequently inserted into hydrogenases during the maturation process. / Furthermore, two Ni binding sites were determined in a monomeric HypB. One is the cluster including C92, H93 and C122, the other is composed of H97 and H101. Upon GTP-dependent dimerization, HypB can bind an extra Ni ion. Our results have shown that the C92/H93/C122 is involved in binding the extra Ni ion, and such binding requires both cysteine residues in the reduced form. Since the GTP-induced dimerization of HypB is coupled to bind an extra Ni, so HypB could act as a GTP-mediated switch that regulate one Ni release from the GTP-bound form to the GDP-bound form. / In the future, we will attempt to crystallize AfHypB in complex with GDP, GTP analogue and AfHypA. Availability of good quality crystals will pave way for the structure determination of AfHypA and AfHypA/HypB complex. And the results obtained will provide a better understanding of the mechanism of functional interaction between HypA and HypB and how HypA and HypB play a role in Ni ion delivery for hydrogenase maturation. / The assembly of the [NiFe]-hydrogenases requires incorporation of Ni ions into the enzyme's metallocenter, which process requires the GTPase activity of HypB and HypA. Due to the essential role in assembly of the active site of hydrogenases, the two proteins were defined as hydrogenase maturation factors. To better understand the mechanism of GTP hydrolysis-dependent Ni delivery accomplished by HypB and HypA, our work focuses on the structure-function study of AfHypB from Archaeoglobus fulgidus and the functional interaction between AfHypA and AfHypB. / The intrinsic GTPase activity of AfHypB is very low, suggesting that AfHypB requires a G-protein activating protein (GAP) to activate its GTPase activity. Although AfHypB can interact with AfHypA to form 1:1 heterodimer, our data suggests that AfHypA is not a GAP for AfHypB. In addition, the FRET results showed that AfHypA could serve as a GEF (G-protein exchange factor) to activate the AfHypB from GDP-bound form to GTP-bound form and facilitate the dissociation of AfHypB dimer in the presence of GMPPNP. / Up to now, we have solved the structure of apo-AfHypB by X-ray crystallography. Crystals of AfHypB were grown using the hanging-drop-vapor-diffusion method and diffracted to ∼2.3 A. It belonged to space group P2(1)2(1)2, with unit cell dimensions a=72.49, b=82.33, c=68.66 A, alpha=beta=gamma=90°. Two molecules of AfHypB were found in an asymmetric unit. Structural comparison between the apo-AfHypB and GTP-bound HypB from M jannachii showed that the GTP binding broke the salt-bridge between K43 and D66, and induced conformational changes in the switch I loop and helix-3, which caused the HypB to form dimer and bind an extra Ni ion. The GTP-bound form of HypB was ready for Ni presenting. And GTP hydrolysis could induce the conformational changes of HypB in the switch I loop and helix-3, which dissociate the HypB dimer into the monomeric GDP-bound form. / Li, Ting. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 98-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites.

Box C/D small ribonucleoproteins (box C/D sRNPs) are responsible for the 2’-O-methylation required for the complete maturation of precursor rRNA. Archaeal box C/D sRNPs, like eucarya, are composed of four components: a guide RNA (box C/D sRNA), an RNA binding protein (L7ae), a 2’-O-methyltransferase (Fibrillarin) and a structural protein (Nop5). Here we develop several approaches for studying box C/D sRNP assembly. In particular, we have used pulldown and mobility shift assays to identify box C/D sRNP assembly intermediates (Nop5-aFib and L7ae-sR1). We have also demonstrated that isothermal titration calorimetry (ITC) can be utilized to quantitatively characterize the energetics of formation for the L7ae-sRNA assembly intermediate. / xi, 98 leaves : ill. (some col.) ; 29 cm

Several vital cellular processes are preformed by large ribonucleoprotein (RNP) complexes. In archaeal and eukaryotic cells one example of these essential RNP particles is the box C/D sRNP. In archaea, this complex is responsible for methylation of ribosomal RNA (rRNA) and transfer RNA (tRNA) during their maturation. Archaeal fibrillarin (aFib) is the 2'-O methyltransferase responsible for catalysis by this complex. In this work we have identified the ability of aFib from Sulfolobus acidocaldarius to form dimers at biologically relevant concentrations and the structural determinants essential for this association. Based on our model we have predicted the ability of aFibs to form dimers in different archaeal and eukaryotic species. The ability of aFibs and their eukaryotic homologs to potentially adopt multiple conformations provides insight into the dynamics of the box C/D sRNP complex. As observed in the study of other essential RNP particles, the ability of these complexes to be conformationally diverse is integral to efficient catalysis of their varied substrates. / viii, 74 leaves ; 29 cm.

The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites.

Thesis (Ph.D.) - University of St Andrews, January 2007.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [87]-101).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiv, 200 p.; also includes graphics (some col.). Includes bibliographical references (p. 167-197). Available online via OhioLINK's ETD Center

Six ribosomal protein genes from the sulfur dependent extreme thermophilic archaebacterium Sulfolobus solfataricus were cloned and sequenced. Four of these genes code for proteins that are equivalent to ribosomal proteins L11, L1, L10 and L12 in Escherichia coli. The other two genes code for proteins that have no equivalent in the eubacteria. The product of one of these genes was found to be equivalent to ribosomal proteins L46 from yeast (Leer et al. 1985a) and L39 from rat liver (Lin et al. 1984), while the product of the other gene shows no sequence similarity to any of the ribosomal proteins present in the data base. In Sulfolobus, the genes that code for ribosomal proteins L11, L1, L10 and L12 are organized in the same order as in Escherichia coli, that is 5' L11, L1, L10, L12 3'. The major transcript from this gene cluster was found to be a 2.5 Kb mRNA that contains the four genes. A less abundant transcript containing only the L10 and L12 gene was also detected. Upstream of the transcription initiation sites, sequences that match the consensus sequence for archaebacterial promoters (TTTAT/AA) were found. Transcription termination sites were located within or after pyrimidine rich regions. Three of the ribosomal protein genes start with unusual initiation codons, GTG in the case of the L1 and L10 genes and TTG in the case of the L11 gene. Putative Shine Dalgarno sequences, complementary to the 3' end of Sulfolobus 16S rRNA, were detected in the region surrounding the initiation codon. In some cases (L1 and L10 genes), the initiation codon was found to be part of this sequence. Sequence comparison of the ribosomal proteins from Sulfolobus with those from other organisms, revealed that the Sulfolobus sequences are closer to those from other archaebacteria, thus supporting the existence of the archaebacterial kingdom. Comparison of the sequences of the L10 and L12 proteins from the three kingdoms revealed that the archaebacterial sequences are closer to the eukaryotes. / Graduate

Deep-sea hydrothermal vents are some of the most biologically productive ecosystems on Earth, yet receive little to no input of photosynthetically derived organic matter. The trophic system at hydrothermal vents is based primarily on the reduction-oxidation (redox) of inorganic chemicals by Bacteria and Archaea. However, the distributional patterns of the microorganisms that colonize deep-sea hydrothermal vent deposits and their link to the geologic setting are still not deeply understood. The goal of the studies presented in this thesis was to quantify the abundance, and distribution of major and understudied vent colonizing archaeal groups from globally distributed and geochemically distinct hydrothermal vent fields. The archaeal community composition was analyzed using quantitative PCR with lineage specific functional gene primers that target methanogens, and 16S rRNA gene primers designed or optimized from this study for the Thermococcales, Archaeoglobus, Ignicoccus and marine Nanoarchaeota. Overall, a general relationship was demonstrated between the geochemical differences of the hydrothermal vent fields and the archaeal community structure. The archaeal community assemblage varied dramatically from hydrothermal vents with different vent host rocks along the Mid-Atlantic Ridge and Eastern Lau Spreading Center. In contrast, two vent fields in the East Pacific, 9°N on the EPR and Guaymas Basin that are basalt and basalt-sediment hosted were found to have similar community composition. These observed differences may be driven in part by the metabolically available chemical energy as hydrogen oxidizing lineages of the methanogens and Archaeoglobus were found in higher abundance in the samples from vent field that had a high concentration of end-member hydrogen and the heterotrophic Thermococcales constituted a higher proportion of the archaeal community at the less enriched vent fields. Interestingly, the Nanoarchaeota and the genus of its only confirmed symbiont, Ignicoccus, were found to have an inconsistent proportional relationship, with the Nanoarchaeota comprising a larger proportion of the archaeal community at the ultramafic and fast spreading basalt vent fields and Ignicoccus at the ultra-slow spreading basalt and andesite hosted vent fields. There was also a more localized pattern identified within the hydrothermal vent deposit. The chemosynthetic lineages of the methanogens and Archaeoglobus constituted a higher proportion of the archaeal community in chimney samples compared to Thermococcales that was found in a higher proportion at horizontal flange samples. This archaeal proportional shift could be driven by energetic micro-niches within the vent deposit, as the chemolithotrophic lineages colonize the area closest to the venting source, and the heterotrophic Thermococcales dominate in more mature structures further from the venting source. Quantitative assessments of the archaeal community composition from this study provided added insight into the dynamic geologic influence on the archaeal lineages that colonize deep-sea hydrothermal vents, on a global and local scale.