Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-89).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 114-145).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-119).

Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT. / text

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.