Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item Allosteric activation of the CRISPR-associated transcription factor Csa 3 by cyclic tetra-adenylate (cA 4)(Montana State University - Bozeman, College of Letters & Science, 2020) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin LawrenceThe CRISPR-Cas immune system provides adaptive and heritable immunity to archaea and bacteria to combat viral infection, and is a source of biochemical tools to researchers. This work combines structural biology and biochemical approaches to provide insight into mechanisms prokaryotes use to control the CRISPR-Cas immune system, linking subsystems into a coordinated response. The first structure of S. solfataricus Csa3 determined by Lintner et al. revealed a dimer with a C-terminal wHTH DNA-binding domain and an N-terminal CARF domain with a putative ligand binding site predicted to bind a two-fold symmetric molecule with both negatively charged and hydrophobic/aromatic moieties, such as dinucleoside polyphosphates or nucleic acid molecules. 1 Later, analysis by Topuzlu et al. of the A. fulgidis Csx3 structure containing a 4 base RNA molecule in a binding pocket revealed similarities between Csx3 and the Csa3 CARF domain, and suggested CARF proteins could bind cyclic or pseudosymmetric linear RNA tetranucleotides represented by the ring-shaped RNA density in the Csx3 binding pocket. 2 Functional studies with S. islandicus Csa3 identified that SiCsa3 regulates transcription of acquisition genes (cas1, cas2, and cas4) and several CRISPR loci. 3,4 Additionally, two groups simultaneously showed that the Type III surveillance complexes produce cyclic oligoadenylate messengers, including cyclic tetra-adenylate (cA 4), which allosterically regulate the RNase activity of Csm6 and Csx1, other CARF proteins. 5,6 These advances support the original predictions by Lintner et al. and suggest that Csa3 binds a cyclic RNA as proposed by Topuzlu et al. Binding of cA 4 likely allosterically causes conformation changes in the wHTH, and regulates the protein's transcriptional regulation. We present the crystal structure of S. solfataricus Csa3 complexed with cyclic tetraadenylate (cA 4). cA 4, as predicted, 1 is bound in the CARF domain 2-fold symmetric pocket, which stimulates conformational changes in the C-terminal domain. Additionally, we identify the presence of a palindromic predicted binding motif upstream of the Type I-A(2) acquisition cassette and CRISPR loci C and D, and reveal through EMSA analysis that Csa3 binds dsDNA nonspecifically with high affinity. Finally, ring nuclease activity is not detected in Csa3, suggesting longer term potentiation of the cA 4 in Csa3 than observed for Csx1/Csm6. 5,6